Thymidylate kinase fusions and uses thereof

ABSTRACT

The application relates to a composition comprising: a stably integrating delivery vector; a modified mammalian thymidylate kinase (tmpk) wherein the modified mammalian tmpk increases phosphorylation of a prodrug relative to phosophorylation of the prodrug by wild-type human tmpk; and a detection cassette fused to tmpk. The application also relates to use of these compositions in methods of treatment of diseases such as graft versus host disease and cancer.

FIELD OF THE APPLICATION

The application relates to compositions, vector constructs and isolated virus and systems comprising a vector and modified thymidylate kinase polynucleotides. The compositions are useful in treatment of diseases such as, inherited genetic disorders cancer and graft versus host disease (GVHD).

BACKGROUND OF THE APPLICATION

Integrating viral vectors are a good choice for gene therapy because they offer fairly efficient transduction and consistent long-term gene expression. Much research has been directed towards improving vector design to increase safety and reliability. A promising approach is to establish control over the fate of transduced cells in vivo. Incorporating an effective suicide gene into a therapeutic vector can ensure that any malignant clones arising from deleterious insertion of the vector can be specifically killed. Likewise, such a control schema could be used as an inserted safety component for a variety of stem cell transplantations, reducing teratomas, for example, should these outgrowth events develop as occurred in one very recent study². A suicide gene schema can also be used to control post-transplant complications.

The infusion of donor lymphocytes in allogenic bone marrow transplant (BMT) recipients provides potent antitumor activity to treat recurrent malignancies. One complication, however, is severe GVHD (Graft Versus Host Disease), which is mediated by T cells in the graft. One approach to control GVHD is to employ suicide gene therapy.

Donor T cells mediate both GVHD and a GVL (Graft Versus Leukemia)-effect after allogenic haematopoietic stem cell transplantation (HCT), and the separation of GVL from GVHD has proven to be a formidable problem. The expression of an inducible suicide gene in donor T cells was conceived as a potential way to provide for the abrogation of GVHD after leukemic cells were eradicated. The most extensively studied suicide genes are derived from pathogens and include the HSV-tk and bacterial cytosine-deaminase genes, which encode enzymes that metabolize ganciclovir and 5-FU, respectively, and generate toxic active compounds (Carolina Berger, Mary E. Flowers, Edus H. Warren, Stanley R. Riddel. Analysis of transgene-specific immune responses that limit the in vivo persistence of adoptively transferred HSV-TK-modified donor T cells after allogenic hematopoietic cell transplantation. Blood 2006, 107:2294-302.)

In the customary adaptation of this approach, the herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene, combined with the antiviral prodrug ganciclovir (GCV), is used to control GvHD after introduction of this suicide gene into donor T lymphocytes. However, the efficiency of HSV1-tk is suboptimal and the issue of host immunogenicity against this heterologous effector gene product can hamper outcomes. In addition, prophylactic GCV is often used to control cytomegalovirus infection after BMT. This confounds the broad clinical implementation of this approach.

HSV1-tk mediated cell killing requires cellular proliferation for its cytotoxic effect. This limits the effectiveness of gene therapies employing tk to only dividing cells. Quiescent disease cells will escape destruction and may persist. Tumor cells have been shown to remain quiescent for long periods of time (Trends Cell Biol. 15(9):494-501, 2005).

SUMMARY OF THE APPLICATION

The inventors' novel cell fate control or suicide gene therapy strategy combines the use of human thymidylate monophosphate kinase (tmpk) fused to a detection cassette such as a CD19 or fluorescent molecule, in a delivery vector such as a lentiviral vector (LV) format and the prodrug Zidovudine (AZT). Since tmpk is endogenously expressed in human cells, immunogenic responses will be limited. This is also true when the detection cassette is human, for example human CD19 or human CD19Δ

The application provides in one aspect a fusion polypeptide comprising:

-   -   (i) a modified mammalian thymidylate kinase (tmpk) wherein the         modified mammalian tmpk increases phosphorylation of a prodrug         relative to phosophorylation of the prodrug by wild-type         mammalian (eg. human) tmpk; and     -   (i) a detection cassette fused to tmpk         wherein the detection cassette polynucleotidpeptide fused to the         tmpk polynucleotidepeptide is a tmpk/detection cassette fusion         polypeptide.         In an embodiment, the fusion polypeptide is an isolated         polypeptide.

In another aspect, the application provides a fusion polynucleotide comprising:

-   -   (i) a modified mammalian thymidylate kinase (tmpk)         polynucleotide wherein the modified mammalian tmpk         polynucleotide encodes a modified mammalian tmpk polypeptide         that increases phosphorylation of a prodrug relative to         phosophorylation of the prodrug by wild type mammalian tmpk         polypeptide; and     -   (ii) a detection cassette polynucleotide fused to the tmpk         polynucleotide;         wherein the detection cassette polynucleotide fused to the tmpk         polynucleotide is a tmpk/detection cassette fusion         polynucleotide.         In an embodiment, the fusion polynucleotide is an isolated         fusion polynucleotide.

In another aspect, the application provides a vector construct comprising:

-   -   (i) a promoter functional in a mammalian cell;     -   (ii) a modified mammalian thymidylate kinase (tmpk)         polynucleotide wherein the modified mammalian tmpk         polynucleotide encodes a modified mammalian tmpk polypeptide         that increases phosphorylation of a prodrug relative to         phosophorylation of the prodrug by wild type mammalian tmpk         polypeptide;     -   (iii) a detection cassette polynucleotide fused to the tmpk         polynucleotide;         wherein the detection cassette polynucleotide fused to the tmpk         polynucleotide is a tmpk/detection cassette fusion and wherein         the coding sequence of the tmpk/detection cassette fusion is         operably connected to the promoter.

The application provides in another aspect a vector construct comprising:

-   -   (i) a stably integrating delivery vector;     -   (ii) a modified mammalian thymidylate kinase (tmpk) wherein the         modified mammalian tmpk increases phosphorylation of a prodrug         relative to phosophorylation of the prodrug by wild-type         mammalian (eg. human) tmpk; and     -   (iii) a detection cassette fused to tmpk         wherein the detection cassette fused to the tmpk is a         tmpk/detection cassette fusion.

A further aspect provides an isolated cell that expresses a modified tmpk/detection cassette fusion.

A further aspect provides an actuable cell destruction component of an expression vector comprising:

-   -   (i) a modified mammalian tmpk polynucleotide wherein the         modified mammalian tmpk polynucleotide increases phosphorlyation         of a prodrug relative to phosphorylation of the prodrug by wild         type mammalian tmpk;     -   (ii) a detection cassette fused to tmpk;     -   (iii) optionally comprising a therapeutic polynucleotide for         expression

Yet a a further aspect provides an isolated virus comprising a viral genome that encodes:

-   -   a modified mammalian thymidylate kinase (tmpk) polynucleotide         wherein the modified mammalian tmpk polynucleotide encodes a         modified mammalian tmpk polypeptide that increases         phosphorylation of a prodrug relative to phosophorylation of the         prodrug by wild type mammalian tmpk polypeptide;     -   a detection cassette polynucleotide fused to the tmpk         polynucleotide;         wherein the isolated virus is infective for a mammalian cell.

Also provided is a cell fate control system comprising:

-   -   a composition, vector construct, or isolated virus for         expressing a tmpk/detection cassette fusion;         wherein a cell expressing the tmpk/dectection cassette fusion is         susceptible to being killed by a prodrug toxic to the cell         expressing the tmpk/dectection cassette fusion. In this system,         the prodrug is administered only if it is desired that the         tmpk/detection cassette expressing cells are to be destroyed         e.g. killed.         Optionally, the cell fate control system further comprises a         prodrug toxic to cells expressing the modified tmpk fused to a         detection cassette.

As the detection cassette is fused to tmpk, permissible cells transfected or transduced with the construct will express tmpk and the detection cassette. This is useful for a number of applications including ensuring that all cells isolated using the detection cassette express the tmpk safety component. A detection cassette fused to tmpk (e.g components (ii) and (iii)) are alternatively referred to as tmpk/detection cassette fusion.

In another aspect the application applicationprovides a composition comprising:

-   -   (i) a stably integrating delivery vector;     -   (ii) a modified mammalian thymidylate kinase (tmpk) wherein the         modified mammalian tmpk increases phosphorylation of a prodrug         relative to phosophorylation of the prodrug by wild-type         mammalian (eg. human) tmpk;     -   (iii) a detection cassette fused to tmpk         wherein the detection cassette fused to the tmpk polynucleotide         is a tmpk/detection cassette fusion.

In an embodiment, the tmpk/dection cassette fusion is a tmpk/dection cassette fusion polynucleotide. The tmpk/dection cassette fusion polypeptide encodes a tmpk/dection cassette fusion polypeptide. Optionally the modified mammalian tmpk/detection cassette fusion polypeptide increases phosphorylation of a prodrug relative to phosophorylation of the prodrug by the wild type mammalian tmpk and/or tmpk/detection cassette fusion polypeptides. Optionally, increased phosphorylation can be determined in a side by side phosphorlyation assay comparing modified mammalian tmpk to wild-type mammalian (eg. human) tmpk.

In one embodiment, the application relates to a composition comprising:

-   -   (i) a stably integrating delivery vector;     -   (ii) a modified mammalian thymidylate kinase (tmpk)         polynucleotide wherein the modified mammalian tmpk         polynucleotide encodes a modified mammalian tmpk polypeptide         that increases phosphorylation of a prodrug relative to         phosophorylation of the prodrug by wild type mammalian tmpk         polypeptide;     -   (iii) a detection cassette polynucleotide fused to tmpk         polynucleotide.

A detection cassette polynucleotide fused to tmpk (e.g components (ii) and (iii) are alternatively referred to as tmpk/detection cassette fusion polynucleotide. The tmpk polynucleotide optionally comprises a polynucleotide with at least 80% sequence identity to a modified tmpk polynucleotide of any one of SEQ ID NOS: 15, 21, and 22. The modified mammalian tmpk polynucleotide optionally comprises a modified human tmpk polynucleotide. The polynucleotide optionally comprises a human polynucleotide and the polypeptides optionally comprise human polypeptides. The modified mammalian tmpk optionally comprises a truncated mammalian tmpk. The modified mammalian tmpk polynucleotide optionally comprises a mammalian tmpk polynucleotide with a point mutation. The point mutation optionally comprises a mutation in a codon of the polynucleotide selected from the group consisting of a mutation that encodes a F to Y mutation at amino acid position 105 (SEQ ID NO: 21), a mutation that encodes a R to G point mutation at amino acid position 16 (SEQ ID NO: 22), and a mutation that encodes a R to A mutation at amino acid position 200 (SEQ ID NO: 16). The polynucleotide optionally further comprises all or part of the large lid or small lid domain of E. coli (SEQ ID NO: 17). It will be readily apparent that one could make 2 or 3 or more amino acid changes and/or combine various mutations. For example F105Y (SEQ ID NO:21) can be combined with R200A (SEQ ID NO: 22). For example, other mutations are readily modeled and derived from the crystal structure of tmpk. Mutations are optionally designed that are inert relative to the active site of the enzyme. The tmpk polynucleotide optionally further comprises all or part of the large lid or small lid domain of E. coli (SEQ ID NO: 17). It will be readily apparent that all or part of large lid or small lid domains from other species of bacteria as well as other organisms such as yeast are useful. Utility is readily established by determining if the large lid or small lid from other sources increases phosphorylation of a prodrug relative to phosophorylation of the prodrug by wild type mammalian tmpk polypeptide. In an embodiment, the tmpk comprises tmpkF105YR200A.

The modified mammalian tmpk optionally comprises one or more deletions. The modified mammalian tmpk polynucleotide optionally has been modified by substituting a portion of wild type tmpk polynucleotide sequence with an exogenous polynucleotide sequence. The substituted portion comprises all or part of a large lid or small lid domain, for example, from E. coli. The exogenous sequence optionally comprises all or part of a bacterial sequence, optionally all or part of a bacterial small lid or large lid domain sequence, optionally an E. coli sequence, optionally TPEVGLKRARARGEL (SEQ ID NO: 17). The small lid domain optionally comprises all or part of amino acids AFGH corresponding to positions 145-148 of human tmpk of SEQ ID NO: 1. The exogenous sequence optionally comprises all or part of a bacterial sequence, optionally all or part of a bacterial small lid sequence, optionally an E. coli sequence, optionally all or part of the amino acid sequence RARGEL corresponding to positions 151-156 of SEQ ID NO: 17. The various mutations, substituions, deletions can be combined. For example, F105Y and R200A can be combined with a substitution and/or deletion described herein. Other combinations are also contemplated. In addition, other mutations not herein disclosed may be combined with the aforementioned mutations.

The detection cassette which is fused to tmpk is optionally selected from the group consisting of CD19, truncated CD19, EGFP, CD25, LNGFR, truncated LNGFR, CD24 truncated CD34, EpoR, HSA and CD20. The detection cassette polynucleotide optionally comprises a truncated CD19 polynucleotide (for example Genbank Accession No. M84371 for full length CD19). For example in one embodiment the truncated CD19 polynucleotide comprises a CD19 polynucleotide truncated at base 939 of CD19 resulting in deletion of the cytoplasmic region of the protein. The polynucleotide optionally comprises a human polynucleotide and the polypeptides optionally comprise human polypeptides. Truncated CD19 is alternatively referred to as CD19Δ or ΔCD19. In an embodiment the truncated CD19 polynucleotide encodes all or part of the amino acid sequence of SEQ ID NO: 28, 29, 31 or 37, optionally encoding SEQ ID NO:37 and/or is optionally the polynucleotide of SEQ ID NO:35.

The tmpk/detection cassette fusion optionally comprises a linker. Optionally, the detection cassette is fused to the tmpk polynucleotide by a linker. The linker in an embodiment encodes all or part of an amino acid sequence of SEQ ID NO:32. The linker, in an embodiment, comprises all or part of the nucleotide sequence of SEQ ID NO:33.

Another aspect provides a composition, vector construct or isolated virus wherein the detection cassette is fused to the tmpk polynucleotide by a linker, where in the linker optionally codes for the amino acid sequence of SEQ ID NO:32. In an embodiment, the composition, vector construct or isolated virus comprises the sequence of the tmpk/detection cassette fusion is the sequence of SEQ ID NO:38, wherein T is optionally substituted with U.

The integrating viral vector optionally comprises a promoter operably linked to the detection cassette polynucleotide and/or the tmpk/detection cassette fusion.

The composition, vector constuct, isolated virus, isolated cell, actuable cell destruction component, or cell fate control system, optionally further comprises a therapeutic polynucleotide cassette.

In an embodiment, the therapeutic polynucleotide cassette is selected from the group consisting of adenosine deaminase, γc interleukin receptor subunit, α-galactosidase A, codon optimized α-galactosidase A, acid ceramidase, galactocerebrosidase, and CFTR molecules.

The integrating delivery vector is optionally selected from the group comprising a retroviral vector, an adenoviral vector, an adeno-associated viral vector, spumaviral, a lentiviral vector and a plasmid or other vector, such as transposons, described in the application. The retroviral vector optionally comprises an oncoretroviral vector. The retroviral vector optionally comprises a lentiviral vector. The vector is optionally a lentiviral vector that has a pHR′ backbone and comprises 5′-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5′-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3′-splice site (SA), Elongation factor (EF) 1-alpha promoter and 3′-Self inactivating LTR (SIN-LTR). Optionally, one makes vectors with the CMV promoter. The lentiviral vector optionally comprises a central polypurine tract (cPPT; SEQ ID NO: 18) and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; SEQ ID NO: 19), optionally the polypurine tract comprises nucleotide nos. 2023 to 2140 and the woodchuck hepatitis virus post-transcriptional regulatory element comprises nucleotide nos. 5802 to 6393 of (SEQ ID NO: 13 or the corresponding nucleotide numbers in SEQ ID NO:14); in a variation, optionally the vector comprises sequences comprising at least 70% sequence identity to one of the foregoing sequences. The lentiviral vector optionally comprises the nucleotides corresponding to the vector backbone portions of SEQ ID NO:13 or SEQ ID NO:14 or the vector backbones pHR′, pDY or pCCL. The composition, vector constuct, isolated virus, isolated cell, actuable cell destruction component, or cell fate control system optionally further comprises an additional kinase wherein the additional kinase contributes to activation of the prodrug. The compositions, polypeptides, polynucleotides, vector constuct, isolated virus, isolated cell, actuable cell destruction component, or cell fate control system of the application are optionally combined with a carrier and form a pharmaceutical composition.

The application also optionally relates to use of these compositions vector constuct, isolated virus, isolated cell, actuable cell destruction component, or cell fate control system in methods of treatment of diseases such as inherited genetic diseases, graft versus host disease and cancer.

applicationAnother aspect optionally provides a method of killing mammalian cells expressing a modified mammalian thymidylate kinase/detection cassette fusion.

In an embodiment, the method comprises:

-   -   i) contacting the mammalian cells with a composition, vector         construct or isolated virus of the application;     -   ii) optionally isolating and/or purifying the cells; and     -   iii) contacting the cells with a prodrug, such as AZT.

Another embodiment of the application relates to a method of killing mammalian cells expressing a modified mammalian thymidylate kinase/detection cassette fusion, comprising:

-   -   i) contacting the mammalian cells with a composition, vector         construct or isolated virus of the application;     -   ii) optionally isolating and/or purifying the cells;     -   iii) transplanting the cells into a transplant recipient; and     -   iv) administering a prodrug to the transplant recipient wherein         the prodrug kills the cells.

It is another object of the application to optionally provide a method of killing mammalian cells expressing a modified mammalian thymidylate kinase/detection cassette fusion comprising:

-   -   i) contacting mammalian cells with a composition of the         application to produce modified cells expressing a modified         mammalian thymidylate kinase/detection cassette fusion         polypeptide;     -   ii) optionally isolating and/or purifying said modified cells;         and     -   iii) contacting said modified cells with a prodrug, such as AZT.

Another embodiment of the application relates to a method of killing mammalian cells expressing a modified mammalian thymidylate kinase/detection cassette fusion polynucleotide, comprising:

-   -   i) contacting the mammalian cells with a composition of the         application to produce modified cells expressing a modified         mammalian thymidylate kinase/detection cassette fusion         polypeptide;     -   ii) isolating and/or purifying said modified cells;     -   iii) transplanting said modified cells into a transplant         recipient; and     -   iv) administering a prodrug to the transplant recipient wherein         the prodrug kills the modified cells.

In another embodiment, the application relates to a method of transplanting cells into a subject comprising administering mammalian cells of the application expressing modified mammalian tmpk/detection cassette fusion (preferably human cells expressing modified tmpk) or other suitable polynucleotide described herein, to the subject.

The application also relates to a method of treating a transplant recipient exhibiting symptoms of a transplant mediated disease comprising administering a prodrug to the transplant recipient. The modified tmpk/detection cassette fusion activates a prodrug by phosphorylation and the activated drug kills the modified tmpk/detection cassette fusion-transduced cells. The method optionally further comprises detecting the presence of the mammalian cells in said transplant recipient one or more times during treatment. Graft versus host disease is an example of a transplant-mediated disease.

The application also optionally relates to a suicide gene safety system for killing a genetically modified cell, the system comprising a construct comprising a suicide gene, such as modified mammalian tmpk/detection cassette fusion polynucleotide or other suitable polynucleotide described herein, capable of activating a prodrug and a polynucleotide of interest to be expressed in said genetically modified cell. A polynucleotide of interest optionally includes a therapeutic molecule. Therapeutic molecules optionally include a normal gene, toxic molecules, cell growth enhancing molecules, or anti-sense molecules. Examples of therapeutic molecules of interest are described in this application, for example therapeutic molecules for treating Fabry disease.

The application also includes a method of expressing a modified mammalian tmpk/detection cassette fusion polynucleotide in a mammalian cell comprising contacting the mammalian cell with a composition, vector construct, isolated virus or cell fate control system of the application.

The contacting is optionally in vitro or ex vivo.

The mammalian cells are optionally selected from the group consisting of stem cells, such as an embryonic stem cell, a mesenchymal stem cell, an induced pluripotent stem (IPS) cell, hematopoietic cells, T cells and human cells. In an embodiment, the cell is a cell from a transplant patient, optionally a bone marrow transplant patient. In an embodiment, the cell is a bone marrow cell.

The mammalian cell is optionally a tumor cell. The tumor cell is optionally contacted with the composition in vivo, for example, using a method selected from the group consisting of microinjection, in vivo electroporation and liposome based methods. The method optionally further comprises administering an effective amount of a prodrug to eradicate the tumor cell. The prodrug optionally comprises AZT. The cells are optionally contacted using a method selected from the group consisting of transfection, transduction, infection and electroporation. The method optionally further comprises isolating the cells. The mammalian cells are optionally isolated by contacting the cells with an antibody that binds to the detection cassette polypeptide wherein the detection cassette polypeptide is selected from the group consisting of CD19, truncated CD19, EGFP, CD25, LNGFR, truncated LNGFR, CD24, truncated CD34, EpoR, HSA and CD20. In a preferred embodiment the detection cassette comprise CD19 or truncated CD19, for example truncated at amino acid 313. The method optionally further comprises a step wherein the isolated mammalian cells are transplanted into a mammal. The mammalian cells are optionally transplanted to a subject, for example to mediate tumor regression.

Another aspect provides a method of expressing a modified mammalian tmpk/detection cassette fusion polypeptide in a subject comprising:

-   -   administering a composition, vector construct, or isolated virus         to the subject;         wherein administration of the composition, vector construct, or         isolated virus results in expression of the modified tmpk         polynucleotide.

Another aspect of the application relates to a method of killing mammalian cells expressing a modified mammalian tmpk/detection cassette fusion polynucleotide comprising:

-   -   contacting the mammalian cells with a composition, vector         construct or isolated virus of the application;     -   optionally isolating the cells; and     -   contacting the cells with an effective amount of a prodrug to         kill the cells.

Another embodiment of the application relates to a method of killing mammalian cells expressing a thymidylate kinase/detection cassette fusion polynucleotide comprising:

-   -   (i) contacting the mammalian cells with a composition vector         construct or isolated virus of the application;     -   (ii) isolating the cells;     -   (iii) transplanting the isolated cells into a transplant         recipient; and     -   (iv) administering an effective amount of a prodrug to the         transplant recipient to kill the transplanted, isolated cells.

The prodrug optionally comprises a substrate that is phosphorylated by a thymidylate kinase polypeptide. The prodrug is optionally selected from the group consisting of thymidine analog, uracil analog, AZT, dT4 and 5-FU.

The mammalian cells expressing said thymidylate kinase polynucleotide are optionally isolated by contacting the cells with an antibody that binds to a detection cassette protein wherein the detection cassette protein is optionally selected from the group consisting of CD19, truncated CD19, EGFP, CD25, LNGFR, truncated LNGFR, CD24, truncated CD34, EpoR, HSA and CD20. The transplant recipient is typically a human and, in certain embodiments, the transplant recipient has, or exhibits, symptoms of graft versus host disease.

Yet a further aspect provides a method of killing a mammalian cell in a subject expressing a modified mammalian tmpk/detection cassette fusion polypeptide comprising:

-   -   administering a composition, vector construct or isolated virus         described herein and/or a cell contacted with the composition,         vector construct or isolated virus; and     -   administering an effective amount of a prodrug to kill the cell.

Another aspect of the application relates to a cell fate control or suicide gene system comprising:

-   -   (i) a stably integrating delivery vector;     -   (ii) a modified mammalian tmpk wherein the modified mammalian         tmpk increases phosphorylation of a prodrug relative to         phosophorylation of the prodrug by wild type human tmpk;     -   (iii) a detection cassette fused to tmpk; and     -   (iv) a prodrug that is phosphorylated and activated by the         modified mammalian tmpk.

Another aspect of the application relates to a suicide gene vector construct comprising a modified mammalian tmpk a detection cassette. The vector construct optionally further comprises a therapeutic cassette. The therapeutic cassette is optionally under the control of a tissue specific promoter and/or an inducible promoter.

Another aspect of the invetion relates to an actuable cell destruction component of an expression vector construct comprising:

-   -   (i) a modified mammalian tmpk polynucleotide wherein the         modified mammalian tmpk polynucleotide increases phosphorlyation         of a prodrug relative to phosphorylation of the prodrug by wild         type mammalian tmpk;     -   (ii) a detection cassette polynucleotide fused to tmpk; and     -   (iii) optionally comprising a therapeutic polynucleotide for         expression.

In the actuable cell fate control component comprising a therapeutic polynucleotide, the therapeutic polynucleotide is optionally selected from the group comprising: adenosine deaminase, γc interleukin receptor subunit, α-galactosidase A, codon optimized α-galactosidase A, acid ceramidase, galactocerebrosidase, and CFTR molecules.

Another aspect of the application relates to a method of killing a cell expressing a modified tmpk/detection cassette fusion polynucleotide comprising contacting the cell with a prodrug that is activated by a composition of the application. The prodrug is optionally a thymidine analog, such as AZT. The modified tmpk polynucleotide is optionally selected from the group comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15; SEQ ID NO: 16, SEQ ID NO: 21 and SEQ ID NO: 22. A detection cassette is polynucleotide is optionally fused to a modified tmpk polynucleotide wherein the tmpk polynucleotide is optionally selected from the group comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15; SEQ ID NO: 16, SEQ ID NO: 21 and SEQ ID NO: 22.

Another aspect of the application relates to a method of killing a cell expressing a modified tmpk/detection cassette fusion polynucleotide in a transplant recipient comprising administering an effective amount of a prodrug that is activated by the modified tmpk polynucleotide. The prodrug is optionally a thymidine analog, such as AZT. In one embodiment, the transplant recipient developed a transplant related adverse event, such as graft versus host disease.

Another aspect of the application relates to a method of reducing cell proliferation, such as treating cancer, in a mammal in need thereof comprising:

-   -   contacting a mammalian cell with a composition of the         application to produce modified cells expressing the modified         mammalian tmpk/detection cassette fusion;     -   isolating the modified cells; and     -   transplanting said modified cells in the mammal wherein the         modified cells induce a graft versus cancer effect.

The method optionally further comprises determining if the transplanted cells induce symptoms of graft versus host disease in the transplant recipient. The method optionally further comprises administering an effective amount of a prodrug to a transplant recipient who exhibits symptoms of graft versus host disease. In a variation, the cancer is leukemia.

Another aspect provides use of a composition, vector construct or isolated virus for expressing a modified mammalian tmpk/detection cassette fusion polypeptide in a mammalian cell or subject.

A further aspect provides use of a composition vector construct or isolated virus for gene therapy.

Yet a further aspect provides use of a composition, vector construct or isolated virus for treating a disease selected from the group consisting of cancer, GVHD or inherited genetic diseases eg diseases resulting from a deficiency of a gene product, optionally Fabry disease.

Also provided is use of an effective amount of a prodrug for killing a cell expressing a modified mammalian tmpk/detection cassette fusion polynucleotide wherein the expression of the modified mammalian tmpk results from contact with a composition vector construct or isolated virus described herein.

Furthermore the application provides compositions, vector contructs, actuable cell destruction components the isolated virus for use as a pharmaceutical, for treating cancer, for treating GVHD, or for treating an inherited genetic disease or disorder.

A further embodiment provides a polynucleotide comprising COalphaGalA (SEQ ID NO:46) wherein T is optionally substituted with U and/or a sequence with at least 95% identity to SEQ ID NO:46, wherein the sequence is not identical to the polynucleotide having accession number NM_(—)000169.

Another embodiment of the application relates to a method of identifying novel thymidine and uracil analog compounds that are useful as prodrugs in combination with a modified tmpk/detection cassette fusion molecule comprising determining if a thymidine or uracil analog is phosphorylated by the modified tmpk/detection cassette fusion molecule. Optionally the determining step comprises, a cell based assay comprising the steps of:

-   -   i) introducing a modified tmpk/detection cassette fusion         molecule into a cell;     -   ii) providing a thymidine analog; and     -   iii) determining whether said thymidine analog is a substrate         for said modified tmpk.

The determining step optionally comprises a cell free assay comprising the steps of:

-   -   i) providing an enzymatically active modified tmpk/detection         cassette fusion,     -   ii) providing a thymidine analog;     -   iii) determining whether said thymidine analog is a substrate         for said modified tmpk/detection cassette fusion.

Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the application are given by way of illustration only, since various changes and modifications within the scope of the application will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the application will be described in relation to the drawings in which:

FIG. 1 is a schematic diagram of recombinant lentiviral transfer vector constructs. A. pHR′-tmpk-IRES-hCD19 is a schematic diagram of a lentiviral vector used to express wild-type tmpk, and mutants F105Y, R-16G-large lid and R200A in combination with a truncated CD19 detection molecule. B. pHR′-IRES-hCD19 is a schematic diagram of a lentiviral vector used to express a truncated CD19 detection molecule. C. pHR′-EGFP is a schematic diagram of a lentiviral vector used to express an EGFP detection molecule. The vector elements illustrated are: LTR—long terminal repeat; ψ—HIV packaging signal, SD—5′ splice signal, RRE—Rev responsive element; SA—3′ splice site, cPPT—central polypurine tract, EF1α—elongation factor 1α promoter; WPRE woodchuck hepatitis virus post-transcriptional regulatory element; SIN—self-inactivating LTR.

FIG. 2 shows a Western blot analysis of tmpk-overexpression by LV-transduction in Jurkat cells. NT: Non-transduced Jurkat cells, IRES: LV-IRES-hCD19-transduced Jurkat cells, WT: LV-(tmpk wild-type)-IRES-hCD19-transduced Jurkat cells, LL: LV-tmpk (R16G, Large lid)-IRES-hCD19-transduced Jurkat cells, F105Y: LV-tmpk (F105Y)-IRES-hCD19-transduced Jurkat cells.

FIG. 3 is a series of graphs comparing transduction efficiencies and hCD19 expression levels in LV-transduced Jurkat cells. Percentages indicate EGFP or CD19 expression and mean fluorescence intensity (MFI) values indicate the levels of expression levels in the cells.

FIG. 4A is a graph illustrating the AZT-sensitivity of Jurkat cells (human T cell line) transduced with LV-tmpk-IRES-hCD19 and mutant tmpk forms. Cell viability was determined by MTT assay (Promega). **, P<0.01, n=3. Data are expressed as mean±standard error of mean (SEM).

FIG. 4B is a series of plots showing annexinV staining.

FIG. 5 is a graph illustrating the induction of apoptosis by the addition of 100 μM AZT in LV-tmpk-transduced Jurkat cells. Cells were seeded in 24 well plates (10⁶/well) in 1 ml of medium with or without 100 μM of AZT. The medium was changed daily. After 4 days of culture, induction of apoptosis in the cells was analyzed by annexin-V staining according to the manufacturer's protocol (Annexin V-APC: BD Pharmingen). **, P<0.01, n=3. Data are expressed as mean±SEM.

FIG. 6A is a graph showing the levels of AZT-metabolites in the cells treated with 100 μM AZT. The cells were cultured in the presence of 100 μM AZT for 36 hrs. 10⁷ cells were homogenized by sonication in 100 ml of 5% (w/v) trichloroacetic acid. The supernatant is collected after homogenate had been centrifuged at 10,000×g for 15 min at 4° C. The trichloroacetic acid was removed by extraction with an equal volume of 20% tri-n-octylamine in pentane. The neutralized aqueous fraction is directly injected into HPLC. Separation of AZT and its metabolites was performed on a C18 column (Waters, Milford Mass.) with a mobile phase composed of 0.2 M phosphate buffer containing 4 mM tetrabutylammonium hydrogen sulfate (pH 7.5) and acetonitrile in the ratio of 97:3 (v/v). The mobile phase was pumped at a flow rate of 1.5 ml/min. The UV absorbance was monitored at 270 nm. Five million cell equivalents were injected and analyzed in triplicate.

FIG. 6B Determination of AZT metabolites in transduced clonal Jurkat cell lines and controls treated with 100 μM AZT. (a) Representative chromatograms for the NT cells and the tmpk R16G-mutant expressing cells. Each arrow indicates the position of a peak of the standard for AZT-MP, AZT-DP, and AZT-TP, respectively. (b) Comparison of the ratio of the intracellular AZT-TP to AZT-MP in the AZT-treated cells. Data are mean±SEM, n=3. The statistical differences were evaluated by the one-way analysis of variance (ANOVA) followed by a Bonferroni post-hoc test with the level of significance set at P<0.05.

FIG. 7 is a graph showing that LV-tmpk-transduced Jurkat cells exhibit an increase in the loss of mitochondrial membrane potential following incubation of the cells with AZT. Cells (10⁶ cells) treated with (shown (+) in figure) or without (−) 100 μM AZT were stained with JC-1 for 15 min at 37° C., and then were analyzed by flow cytometry. ***, P<0.001, n=3.

FIG. 8 is a series of graphs showing that AZT can induce apoptosis in the absence of cellular proliferation. Cellular proliferation is not always a prerequisite for AZT-induced apoptosis. Cells were seeded in 24-well plates (10⁶/well) in 1 ml of medium containing 0 (shown in AZT (−) in figure) or 100 μM of AZT (shown in AZT (+)) with or without 5 μM indirubin-3′-oxime (Figure (B) and (A), respectively). The medium was refreshed daily. After 2 days of culture, induction of apoptosis by AZT was analyzed by annexin V staining according to the manufacturer's protocol described. **, P<0.01, n=3. Data are expressed as mean±SEM.

FIG. 9 is a graph showing that mutant forms of tmpk prevent growth of transduced K562 cells xenografted into AZT-treated NOD/SCID mice. Female or male 5 to 8-week-old non-obese diabetic/sever combined immunodeficient (NOD/SCID) mice were purchased from Jackson Laboratory. Lentivirally-transduced or non-transduced K562 cells (20×10⁶ cells) were resuspended in 0.5 mL Dulbecco's phosphate-buffered saline (D-PBS) per inoculum and injected subcutaneously (SC) into the right flanks of recipient mice. AZT treatment, which was administered intraperitoneally (IP) at the dose of 2.5 mg/kg/day, was started one day after injection and conducted for 14 days. In vivo tumor cell growth was monitored by measuring tumor size for up to 32 days post-inoculations. All experimental data were reproduced at least twice.

FIG. 10 is a series of graphs evaluating the transduction efficiencies in the infected primary human T cells by detecting the transgene expression 6 days after transduction. 1-3: Transgene expression in primary human T cells transduced with 1:LV-EGFP, 2: LV-(tmpk R-16GLL)-IRES-hCD19, 3: LV-IRES-hCD19 Condition A-No transduction, Condition B-Single transduction using fibronectin (FN), Condition C-Three repeated transductions using FN, Condition D-Single transduction without FN, Condition E-Three transductions without FN. The cells are transduced repeatedly every 24 h at the MOI indicated.

FIG. 11 is a graph confirming transgene expression in the primary cultured mouse T cells isolated from spleen. Primary murine splenic T cells were isolated from the spleen of a Balb/c mouse. The cells were cultured for 3 days using anti-CD3/28 beads and 20 IU/ml recombinant human interleukin-2 (rhIL2). Cells were transduced using fibronectin (FN)-coated plates using an MOI of 20. EGFP-expression in the infected cells was confirmed 6 days post-transduction. Data are expressed as mean±SEM. P<0.001, n=3.

FIG. 12 is a graph comparing transgene expression in the cultured primary mouse T cells isolated from the spleen. The activated murine T cells were transduced with LVs indicated in the figure using either an FN-coated plate or transduction-on-ice methods. Transgene expression in the infected cells was confirmed 6 days post-transduction, n=2.

FIG. 13 is a listing of sequences.

FIG. 14 is a graph showing the measurement of AZT sensitivity of clonally-derived Jurkat cells transduced with LV-tmpk-IRES-huCD19Δ and control vectors. Cell viability was measured by MTT assay following 4 days incubation with or without AZT. The results were shown as percentage of the A595 nm value from the assay. The negative control values (without AZT) and the values without cells were deemed as 100% and 0%, respectively. Data are presented as the mean±SEM, n=3. The statistical significance of experimental observation was determined by the one-way ANOVA followed by a Dunnett post-hoc test with the level of significance set at P<0.05 compared with the values of the control group of cells that were not treated with AZT. *, P<0.05, and **, P<0.01 vs. the cells without AZT-treatment in each group.

FIG. 15 shows the induction of apoptosis by addition of 100 μM AZT in clonal Jurkat cells transduced with LV-tmpk-IRES-huCD19Δ and control vectors. Cells were cultured in the absence (A) or presence (B) of 5 μM indirubin-3′-monoxime for 4 days with or without 100 μM AZT. To compare the effect of AZT on induction of apoptosis in each group, measurement of flow cytometric analyses obtained from the cells treated with AZT were normalized by dividing values by those obtained without AZT. Data are mean±SEM, n=3. The statistical differences were evaluated by the one-way ANOVA followed by a Bonferroni post-hoc test with the level of significance set at P<0.05. **, P<0.01 vs. the NT cells.

FIG. 16 shows the transduction of primary murine and human T cells. (A) Observed levels of huCD19Δ expression on primary murine T cells 5 days after cells were transduced a single time with concentrated LV at an MOI of 20. (B) Observed levels of huCD19Δ expression on transduced primary human T cells. (C) Fold increases in the apoptotic index in the presence of 100 μM AZT.

FIG. 17 presents an analysis of the mechanism of induction of apoptosis by AZT in the tmpk-mutant expressing cells. (A) The tmpk mutant expressing cells treated with AZT showed an increase in the loss of mitochondrial membrane potential. Following 4 days incubation with or without 100 μM AZT, cells were stained with JC-1 for 15 min at 37° C., and then were analyzed by flow cytometry. To compare the effect of AZT on the increase in the loss of mitochondrial membrane potential at the day 4, the statistical differences were evaluated by the one-way ANOVA followed by a Bonferroni post-test with the level of significance set at P<0.05. ***, P<0.001, n=3. (B) Activation of caspase 3 in transduced cells by AZT treatment. Cells were cultured for 4 days with or without 100 μM AZT. To compare the effect of AZT on activation of caspase 3 in each group, measurement of flow cytometric analysis obtained from the cells treated with AZT were normalized by dividing those without AZT. Data are mean±SEM, n=3. The statistical differences were evaluated by the one-way ANOVA followed by a Bonferroni post-test with the level of significance set at P<0.05. **, P<0.01 and ***, P<0.001 vs. NT.

FIG. 18 shows that a daily injection of AZT prevents growth of K562 cells transduced with LV-tmpk-mutant in NOD/SCID mice. (A) NOD/SCID mice were subcutaneously injected with 2×10⁷ cells of either the NT or the LV-transduced K562 cells into the dorsal right flank. Starting one day after the cell injection, the mice received daily intraperitoneal injections of AZT (2.5 mg/kg/day) for two weeks. Tumor volume was monitored at the day indicated in the figure. (B) The tumor volume on day 14 (at the end point of the experiment) is shown. Data are mean±SD, n=5. The statistical comparison of means was performed by a two-tailed unpaired Student's t test.

FIG. 19. Ratio of AZT-DP and AZT-TP compared to AZT-MP in 293T cells transfected with pCIneo/CD19ΔTmpkF105YR200A/IRES and a non-transfected control group (NT) determined by HPLC analysis.

FIG. 20. Cell viability of Jurkat cells transduced with LV/CD19ΔTmpkF105YR200A is significantly reduced when incubated in 100 μM and 1000 μM compared to the non-transduced control group. Values in each group are normalized to the 0 μM AZT value, with this value being set to 1.0.

FIG. 21. A plasmid map for the lentivector pCCL.SIN.cPPT.EF. CD19ΔTmpkF105YR200A.WPRE engineering expression of the CD19ΔTmpkF105YR200A fusion protein.

FIG. 22. A plasmid map for pHR′-CD19ΔTmpkF105YR200A.

FIG. 23. Development of Novel CD19/TmpK Fusion ‘Cell Fate Control’ Gene Illustration.

FIG. 24. A graph showing sensitivity of Jurkat Cells Transduced with LV/CD19Tmpk-Fusion Towards AZT

FIG. 25. Development of Bicistronic Lentivirus: CD19/TmpK Fusion with Secondary αGalA

FIG. 26. A graph showing αGalA enzyme activity of transduced Fabry Fibroblast with the LV/ΔCD19/TmpK-IRES-αGalA

FIG. 27. A plasmid map of pDY.CD19deltaTmpK

FIG. 28. A plasmid map of pDY.CD19deltaTmpK-IRES-CO-αGalA

FIG. 29. Is a series of graphs which show K562 Cells transduced at MOI 10. Gated on live cells (7AAP).

FIG. 30. Is made up of two graphs (A and B) which represent: A) Alpha-GalA Enzyme Activity in Fabry Primary Fibroblast and B) Fabry Fibroblast cells. Transduced Fabry Fibroblast with pDY-CD19ΔTmpK-IRES-COαGalA show enzyme correction.

FIG. 31. Is a series of Graphs. A) shows Alpha-GalA Enzyme activity and B) shows FACS analysis of COαGalA transduced cells. Transduced K562 Cells with pDY-CD19ΔTmpK-IRES-COαGalA shows over-expression of αGalA.

DETAILED DESCRIPTION OF THE APPLICATION

The inventors herein present a novel prodrug/enzyme combination for suicide gene therapy also known as cell fate controlled gene therapy, for treating GVHD and therapeutic gene therapy. Catalytically improved variants of human tmpk/detection cassette fusions were delivered into target cells by novel lentiviruses (LVs), and the ability to selectively clear these cells in vitro in response to increasing AZT concentrations was evaluated. The inventors demonstrate the highly efficient transfer of these suicide genes fused to a truncated huCD19 marker into cell lines. AZT sensitivity in transduced cells was further analysed. The inventors additionally demonstrate that increased accumulation of intracellular AZT-TP in tmpk-mutant-transduced cells decreases cell viability. Without wishing to be bound by theory, this is in part due to the activation of a mitochondria-mediated apoptosis pathway. These results show that the rationally designed minimal mutants of tmpk/detection cassette fusions employed are a practical choice for suicide gene therapy and establish the next generation of safer integrating viral vectors. In addition, this system is useful to endow stem cells (both embryonic and of later ontogeny) destined for utility in clinical transplantation, for example, with a reliable safety system. Further the system provides a novel safety feature for therapeutic gene therapy, such as for treating Fabry disease.

Accordingly, the application relates to tmpk/detection cassette fusion polypeptides and nucleic acids, vector constructs, compositions and cells comprising these, and systems employing as well as methods of using said tmpk/detection cassette fusions. For example said tmpk/detection cassette fusion gene mutants can be inserted in transplant cells for treatment of cancer and controlling transplant-associated graft versus host disease. A lentrivirus is optionally used to deliver tmpk/detection cassette fusions. Other methods of delivery are also useful.

The application prodrug/enzyme combination works by increasing phosphorylation of prodrugs such as AZT. For example, the prodrug AZT is converted through a series of phosphorylation steps into AZT-triphosphate (AZT-TP)¹². This is the active metabolite that inhibits replication of the human immunodeficiency virus (HIV)¹³⁻¹⁵, and to a lesser extent, DNA replication in eukaryotic cells¹⁶. Safety profiles for this compound are well known and concentrations of AZT in the bloodstream of AIDS patients being treated with this agent can reach high levels. The rate-limiting step in the conversion of AZT to the toxic AZT-TP form is the intermediate step of phosphorylation of AZT-monophosphate (AZT-MP) to AZT-diphosphate (AZT-DP) catalyzed by the cellular thymidylate kinase (tmpk), which has a low enzymatic efficiency for AZT-MP¹⁷. Accumulation of AZT-metabolites in the cells of AZT-treated AIDS patients reportedly induces toxic mitochondrial myopathy¹⁸⁻²². To harness this dual toxicity of AZT-TP, the inventors developed a novel suicide gene therapy approach based on the engineered overexpression of human tmpk. In order to improve the processing of AZT-MP to AZT-DP, thereby increasing intracellular AZT-TP concentrations, the inventors have engineered minimally modified tmpk mutants (F105Y and R16G-Large lid (RG16GLL)) with approximately 200-fold enhanced activity for AZT-MP^(23,24).

Phosphorylation of the prodrug leads to its activation and increases its effectiveness in killing tmpk transduced cells (also called “suicide gene therapy”).”). As used herein “suicide gene therapy” and “cell fate controlled gene therapy” are used interchangeably.

The application is useful in the event of a transplant related adverse event. A transplant related adverse event typically comprises graft versus host disease where following T-cell (or other cell) transplant to a recipient the transplanted cells attack the host. A transplant adverse event also comprises any situation where it would be beneficial to eliminate the transplanted cells, including where transplanted cells comprise integrations that can cause disease. For example, the transplanted cells express mutant tmpk so that upon detection of graft versus host disease, a prodrug such as AZT is optionally administered to the patient to kill the transplanted cells.

For cancer treatment, the above method is useful to treat leukemia where donor transplant cells are used to kill leukemic cells. The transplanted cells expressing tmpk are likely to also attack the host, so the application allows the transplanted cells to be killed after detection of the onset of graft versus host disease.

In a variation of the application, tmpk/detection cassette fusion vector constructs are inserted directly into the solid tumor and expression of tmpk/detection cassette fusion sensitizes the cells to the prodrug.

Additionally, the tmpk/detection cassette fusion gene mutants are useful as a general ‘safety component’ in gene therapy. For example, use of the tmpk/detection cassette fusion in combination with alpha-galactosidase is useful for treating patients with Fabry disease. Also, in patients with Severe Combined Immunodeficiency Disease (SCID), gene therapy has been used successfully to introduce deficient genes however at least one clinical trial was halted due to safety concerns arising from inappropriate DNA integrations. The prior art also includes much discussion about the dangers of gene therapy due to vector integrations that can cause cancer. The safety component overcomes this problem by allowing the transplanted cells to be destroyed upon administration of a prodrug.

Tmpk/Detection Cassette Fusion Molecules

One aspect of the application relates to isolated polypeptides and isolated nucleic acids that encode a tmpk/detection cassette fusion.

As used herein “tmpk/detection cassette fusion” refers to a molecule comprising a tmpk component such as a modified tmpk or tmpk variant described herein and a detection cassette component fused in frame with tmpk. Tmpk/detection cassette fusion molecules include, polypeptides, polynucleotides, as well as variants and fragments thereof that maintain detection capacity and tmpk catalytic activity.

The tmpk/detection cassette fusion is optionally constructed such that the tmpk component can be N-terminal (or 5′) or C-terminal (or 3′) in continuous or discontinuous relationship to the detection cassette component. For example, in a continuous relationship the fusion polypeptide can comprise a tmpk component fused to a detection cassette (e.g. NH2-tmpk-GFP-COOH) or alternatively can comprise a detection cassette component fused to a tmpk molecule (e.g NH2-GFP-tmpk-COOH). Similarly, a fusion polynucleotide can comprise a tmpk component fused to a detection cassette (e.g. 5′-tmpkGFP) or alternatively can comprise a detection cassette component fused to a tmpk molecule (e.g 5′-GFP-tmpk-3′)).

In addition the tmpk and detection cassette components are optionally discontinuous. For example a linker sequence is optionally present between the tmpk and detection cassette components.

The term “linker sequence” as used in reference to a tmpk/detection cassette fusion refers to residues that link the tmpk and detection cassette components. In a polypeptide, the residues are generally amino acids. In a polynucleotide, the residues are generally nucleotides. The term “linker sequence” as used in reference to a tmpk/detection cassette fusion polypeptide accordingly generally refers to a sequence of amino acids that link the tmpk and detection cassette components. The term “linker sequence” as used in reference to a tmpk/detection cassette fusion polynucleotide accordingly generally refers to a sequence of nucleotides that link the tmpk and detection cassette components. The linker when referring to a polypeptide sequence optionally comprises 3, 4, 5, 6, 6-10, 10-15 or 15-25 amino acids or longer and when referring to a polynucleotide sequence comprises 3-6, 6-12, 18, 12-24, or 24-72 nucleic acid residues or longer. A linker sequence is useful for several reasons. A linker sequence can be used to facilitate cloning. Further a linker sequence can provide a gap between the components that facilitates proper folding and/or activity (e.g. antigenic activity for the detection cassette and/or catalytic activity for the tmpk component). A person skilled in the art will recognize that a number of linker sequences can be used and a number of linker sequences are known in the art. The linker sequence can comprise any sequence of amino acids or nucleotides that is suitable. For example, suitable refers to the amino acid composition of the linker. For example uncharged amino acids are preferable. Amino acids such as proline which could limit the flexibility of the linker are generally not preferred. In one embodiment of a discontinuous relationship, the fusion polypeptide optionally comprises a tmpk component fused to a linker fused to a detection cassette (e.g. NH2-tmpk-linker-GFP-COOH or alternatively comprises a detection cassette component fused to a linker fused to a tmpk molecule (e.g NH2-truncated CD19-linker-tmpk-COOH). Similarly, a fusion polynucleotide can comprise a tmpk component fused to a linker fused to a detection cassette (e.g. 5′-tmpk-linker-GFP-3′) or alternatively can comprise a detection cassette component fused to a linker fused to a tmpk molecule (e.g 5′-truncated CD19-linker-tmpk-3′; such as SEQ ID NO: 28, 29, 31 or 37 fused to a linker sequence described herein fused to SEQ ID NO:36)). The tmpk and detection cassette components are fused in frame such that both components are expressed together as one continuous polypeptide sequence in each cell. The various tmpk and detection cassette components are further described below.

Fusion Polypeptides

As mentioned, the application provides isolated tmpk/detection cassette fusion polypeptides. A tmpk/detection cassette fusion polypeptide comprises a tmpk polypeptide component and a detection cassette polypeptide component.

The term “isolated polypeptide” refers to a polypeptide substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.

As mentioned, tmpk polypeptide components that can be comprised by the tmpk/detection cassette fusion polypeptide are described in detail below. In one embodiment the tmpk polypeptide component comprises TmpkF105YR200A. In a more specific embodiment the tmpk polypeptide component comprises the amino acid sequence in SEQ ID NO:36.

In another preferred embodiment, the detection cassette polypeptide component comprises truncated CD19 polypeptide. In a more specific preferred embodiment, the truncated CD19 polypeptide component comprises the sequence in SEQ ID NO:37.

In another embodiment the tmpk/detection cassette fusion polypeptide comprises a linker sequence, for example amino acids A G G A A G (SEQ ID NO: 32).

In another embodiment, the tmpk/detection cassette fusion polypeptide comprises a truncated CD19, a linker sequence and tmpkF105YR200A. In one embodiment the amino acid sequence comprises the amino acid sequence in SEQ ID NO:38.:

A person skilled in the art will recognize that conservative amino acid substitutions as well as additions/deletions or a number of divergent amino acid sequences can be used are readily made to the disclosed sequences and are within the scope of the present disclosure.

A “conservative amino acid substitution” as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Conservative amino acid substitutions are known in the art. For example, conservative substitutions include substituting an amino acid in one of the following groups for another amino acid in the same group: alanine (A), serine (S), and threonine (T); aspartic acid (D) and glutamic acid (E); asparagine (N) and glutamine (Q); arginine (R) and lysine (L); isoleucine (I), leucine (L), methionine (M), valine (V); and phenylalanine (F), tyrosine (Y), and tryptophan (W).

Fusion Nucleic Acids

The application also provides isolated tmpk/detection cassette polynucleotides. A tmpk/detection cassette fusion polynucleotide comprises a tmpk polynucleotide component and a detection cassette polynucleotide component.

The term “polynucleotide” and/or “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine.

The term “isolated polynucleotide” or “isolated nucleic acid sequences” as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) from which the nucleic acid is derived. The term “nucleic acid” is intended to include DNA and RNA and can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term “nucleic acid” includes the complementary nucleic acid sequences.

As mentioned, tmpk polynucleotide components that can be comprised by the tmpk/detection cassette fusion polynucleotide are described in detail below. In a preferred embodiment, the tmpk polynucleotide component comprises tmpkF105YR200A. In a more specific embodiment the tmpk polynucleotide component comprises the nucleic acid sequence in SEQ ID NO:34.

In another preferred embodiment, the detection cassette polypeptide component comprises truncated CD19 polynucleotide. In a more specific preferred embodiment, the truncated CD19 polynucleotide component comprises the sequence in SEQ ID NO:35.

In another embodiment the tmpk/detection cassette fusion polynucleotide comprises a linker sequence, for example GCCGGCGGGGCTGCAGGG (SEQ ID NO: 33).

In another embodiment, the tmpk/detection cassette fusion polynucleotide comprises a truncated CD19, a linker sequence and tmpkF105YR200A. In one embodiment the polynucleotide sequence comprises the sequence in SEQ ID NO:39.

The tmpk polynucleotide sequence provided above comprises silent mutations compared to the wildtype sequence of tmpk (in addition to the F105YR200A encoding mutations). Base 318 is converted from “C” to “A” and base 345 is converted from “T” to “C”. These mutations do not alter the amino acid sequence of the protein. Accordingly a person skilled in the art will recognize that sequence variants that do not result in an amino acid change are useful and within the scope of the present disclosure. In addition, nucleotide changes that result in conservative changes and/or changes wherein the activity of the components is maintained (e.g. antigenic activity of detection cassette and catalytic activity of the tmpk component).

The application also provides for polynucleotides that bind tmpk/detection cassette polynucleotides and/or their complements under at least moderately stringent hybridization conditions. The polynucleotides that hybridize can comprise polynucleotides with sequence identity with the tmpk, linker, and/or detection cassette and which retain activity. In one embodiment, the polynucleotide that hybridize comprises 60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, 99-99.9% or more sequence identity.

The term “hybridize” refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid.

By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm=81.5° C. -16.6 (Log 10 [Na+])+0.41(% (G+C)−600/l), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5× sodium chloride/sodium citrate (SSC)/5×Denhardt's solution/1.0% SDS at Tm−5° C. based on the above equation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderately stringent hybridization conditions include a washing step in 3×SSC at 42° C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

The term “sequence identity” as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions.times.100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present application. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

Vector Constructs

The aforementioned isolated polynucleotides are optionally comprised in a vector to provide a vector construct, optionally herein referred to as a vector. The vector construct optionally comprises:

-   -   (i) a stably integrating delivery vector;     -   (ii) a modified mammalian thymidylate kinase (tmpk) wherein the         modified mammalian tmpk increases phosphorylation of a prodrug         relative to phosophorylation of the prodrug by wild-type         mammalian (eg. human) tmpk;     -   (iii) a detection cassette fused to tmpk.         Each of these components is further described below.

Tmpk Variants

Thymidylate kinase is a kinase that catalyzes the addition of a phosphoryl group to thymidylate as well as thymidine analogs such as AZT. Several wild-type human sequences have been reported. SEQ ID NOS: 1, 3, 5 and 7 are reported nucleotide sequences of human thymidylate kinase (SEQ ID NO: 7 does not have a stop codon). The different sequences represent natural polymorphic variations present in the population and it will be recognized in the art that future identified molecules with polymorphic variations will also be considered to be wildtype tmpk. SEQ ID NO: 9 is the reported mouse thymidylate kinase sequence. The mouse sequence shares 82% nucleotide identitiy 81% amino acid identity and several residues that have been identified as limiting the nucleoside analog activity of the human tmpk enzyme and which result in increased enzymatic activity when modified, are conserved in the murine sequence. The corresponding amino acid sequences are reported in SEQ ID NOS: 2, 4, 6, 8, and 10. SEQ ID NO: 2 provides the amino acid sequence for the wild-type tmpk polynucleotide described in SEQ ID NO: 1; SEQ ID NO: 4 provides the amino acid sequence for the wild-type tmpk polynucleotide reported in SEQ ID NO: 3, SEQ ID NO: 6 provides the amino acid sequence for the wild-type tmpk polynucleotide described in SEQ ID NO: 5; SEQ ID NO: 8 provides the putative sequence of the wild-type tmpk polynucleotide reported in SEQ ID NO: 7; and SEQ ID NO: 10 provides the amino acid sequence of the wild-type murine tmpk polynucleotide described in SEQ ID NO: 9. Modified tmpk molecules and mutant tmpk refer to mammalian tmpk molecules that have been modified compared to wild-type. Among the mutant tmpks, some of these showed a superior enzymatic activity to convert deoxy-thymidine-monophosphate (dTMP) to dTMP-diphosphate (dTDP) or AZT-MP to AZT-DP. Increased kinase activity relative to wild-type refers to modified tmpk molecules that exhibit improved enzymatic kinetics compared to tmpk wild-type. The improved activity comprises increases in binding and or enzymatic turnover to convert the monophosphate-form of the substrate of tmpk to the diphosphate form.

Mutations which show superior enzymatic activity included the F105Y mutant (SEQ ID NO: 11, SEQ ID NO: 21), R16GLL mutant (SEQ ID NO: 12, SEQ ID NO: 22) and the R200A mutant (SEQ ID NOS: 15 and 16).

One aspect of the application provides vector constructs comprising modified detection cassette fused tmpk enzymes with increased nucleoside analog kinase activity relative to wild-type. In one aspect, the modification that increases tmpk nucleoside analog kinase activity comprises one or more deletions. The deletions can be internal or can result in a truncated variant. In an alternate embodiment the modification that increases tmpk nucleoside analog kinase activity comprises one or more point mutations. In another embodiment an exogenous sequence replaces an endogenous sequence. For example, in one embodiment all or part of the large lid domain of human tmpk (SEQ ID NO:20) is replaced with all or part of the large lid domain of a different species. In one embodiment the different species is a bacteria species. In one embodiment, all or part of the large lid domain of human tmpk (SEQ ID NO:20) is replaced with all or part of the large lid domain of E. coli tmpk (SEQ ID NO:17). In another embodiment, residues 145-148 of SEQ ID NO: 1 (AFGH) are replaced with all or part of the small lid region of E. coli residues 151-156 in SEQ ID NO: 17 (RARGEL). In another embodiment the modified tmpk is selected from the group including the F105Y mutant (SEQ ID NO: 11, SEQ ID NO: 21), R16GLL mutant (SEQ ID NO: 12, SEQ ID NO: 22), a tmpk molecule modified by the substitution of all or part of a bacterial large lid domain such as the E. coli large lid domain in SEQ ID NO: 17, a tmpk molecule modified by the substitution of all or part of a bacterial small lid domain such as the E. coli small lid domain at 151-156 of SEQ ID NO: 17, and the R200A mutant (SEQ ID NOS: 15 and 16).

In another embodiment, the exogenous sequence is optionally synthesized or obtained from a non-mammalian thymidylate kinase such as a bacterial thymidylate kinase. As used herein a modified mammalian tmpk molecule includes a modified tmpk molecule that comprises non-mammalian sequences such as all or part of either a large lid domain or a small lid domain sequence from bacteria such as E. coli. A variant may comprise one or more of the aforementioned modifications. Examples of modifications are described above.

Detection/Selection Cassettes

In cell fate control or suicide gene therapy, it is typically desirable that the majority, and preferably all of transduced cells express the suicide gene. This need can be met by co-introducing a cell surface marker detection gene cassette. Vector constructs wherein the detection cassette and suicide gene are independently translated e.g separated by an IRES sequence, cells may express only one of the two genes. A fusion protein fusing the suicide gene and the detection cassette can be useful to minimize cells expressing only one of the genes. Transduced cells can be identified and enriched based on expression of the fusion protein. A good detection cassette gene should be inert in itself, devoid of signaling capacity and non-immunogenic²⁸. A variety of cell surface markers can be used in this context: human CD24²⁹, murine HSA³⁰, human CD25 (huCD25)³¹ and a truncated form of LNGFR³².

While huCD25 has been an efficient and malleable marker for murine studies^(27,33), it is not useful for gene transfer applications into T cells since expression of this molecule is up-regulated when this population is activated. Other groups have also used the truncated form of LNGFR³², but overexpression of this marker has been reported to promote transformation of myeloid cells in an unusual, highly context-dependent manner³⁵. A novel truncated form of CD19 (e.g. CD19Δ) is adopted in one embodiment as a detection cassette (SEQ ID NOS: 29-31). CD19 (SEQ ID NOS: 27-28) is a 95-kDa glycoprotein of the immunoglobulin superfamily. It forms a complex with CD21, CD81, and Leu-13, and collectively functions to modulate the activation threshold of the B cell receptor^(36,37). As expression of CD19 and CD21 is restricted to B cell lineages from immature progenitors to blasts³⁸, it is suitable for use in murine and human T cells. To further decrease any signaling capacity from the CD19 molecule, the cytoplasmic tail³⁹ has been deleted for the present adaptation. In one embodiment truncated CD19 comprises all or a portion of SEQ ID NO: 29. In another embodiment truncated CD19 comprises all or a portion of SEQ ID NO: 30. In another embodiment truncated CD19 comprises all or a portion of SEQ ID NO: 31.

“Detection cassette” is used to refer to a polynucleotide that directs expression of a molecule that acts as a cell marker and that optionally provides for a mode of isolating cells expressing said marker. The molecule is optionally used to select transduced or transfected cells or to determine the efficiency of cell transduction or transfection. Molecules that are useful as cell markers or detection agents comprise CD19, truncated CD19, CD25 and EGFP, HSA, CD20, GFP, ETC. EGFP is variably referred to as enGFP or GFP herein. One skilled in the art will recognize that other fluorescent molecules can similarly be used. These molecules can be fused to tmpk to provide a tmpk/detection cassette fusion molecule.

The term “fused” as used herein means that the detection cassette and tmpk molecule are in the correct reading frame eg the expression fusion comprises a functional detection cassette and functional tmpk. The tmpk detection cassette can be continuous (e.g no linker) or can be discontinous (e.g comprise a linker).

As mentioned, the detection cassette encodes a molecule that is typically used to isolate transduced or transfected cells. The detection cassette is useful in vectors comprising modified tmpk or control molecules. Control molecules include molecules that do not function as suicide gene therapy molecules which that are typically employed to assess the effect of tmpk mutants in similarly related cells. A person skilled in the art would recognize that many molecules are useful for fusing to tmpk to permit isolation of modified tmpk or control expressing cells. Choice of molecule will depend on the cell type to be transfected or transduced. Generally, the detection cassette molecule is not expressed on the cell type to be transfected or transduced in appreciable levels permitting isolation of cells expressing the detection cassette. In one embodiment the detection cassette encodes a CD19 (SEQ ID NOS: 27-28). In a preferred embodiment, the detection cassette encodes a truncated CD19 (SEQ ID NOS: 29-31). In an alternate embodiment, the detection cassette encodes CD25. In another embodiment, the detection cassette encodes a fluorescent protein such as EGFP. In another embodiment, the molecules encoded by the detection cassette comprise CD20, CD25, low affinity nerve growth factor receptor (LNGFR), truncated CD34, or erythropoietin receptor (EpoR). Additionally, the detection cassette can comprise a drug resistance gene permitting isolation of transduced or transfected cells by drug selection.

Delivery Vectors

As used herein “delivery vector” refers to a nucleic acid molecule that is used as a vehicle to deliver one or more nucleic acid molecules of interest, such as transgenes, into a cell. Delivery vector can refer optionally to the plasmid construct (e.g. vector backbone such as pHR) that is used to generate virus or to a virus genome. Optionally, the delivery vector is constructed to permit expression of one or more transgenes and can be referred to as an expression vector. A delivery vector into which has been inserted one or more nucleic acids to be transferred to a cell, is referred to as a vector construct.

It will be appreciated by one skilled in the art that a variety of delivery vectors (e.g. vector backbones) and expression vehicles are usefully employed to introduce a modified tmpk/detection cassette fusion molecule into a cell. Delivery vectors that are useful comprise lentiviruses, oncoretroviruses, expression plasmids, adenovirus, and adeno-associated virus. Other delivery vectors that are useful comprise herpes simplex viruses, transposons, vaccinia viruses, human papilloma virus, Simian immunodeficiency viruses, HTLV, human foamy virus and variants thereof. Further vectors that are useful comprise spumaviruses, mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, mammalian type D retroviruses, HTLV/BLV type retroviruses, and lentiviruses.

Delivery vectors such as those listed above have been employed to introduce thymidine kinase molecules into cells for use in gene therapy. Examples of delivery vectors used to express thymidine kinase in cells include: Kanazawa T, Mizukami H, Okada T, Hanazono Y, Kume A, Nishino H, Takeuchi K, Kitamura K, Ichimura K, Ozawa K. Suicide gene therapy using AAV-HSVtk/ganciclovir in combination with irradiation results in regression of human head and neck cancer xenografts in nude mice. Gene Ther. 2003 January; 10(1):51-8. Fukui T, Hayashi Y, Kagami H, Yamamoto N, Fukuhara H, Tohnai I, Ueda M, Mizuno M, Yoshida J Suicide gene therapy for human oral squamous cell carcinoma cell lines with adeno-associated virus vector. Oral Oncol. 2001 April; 37(3):211-5.

The term “stably integrating” as used herein refers to components that permit the delivery construct and its inserts (eg modified tmpk/detection cassette fusion) to be maintained in a cell. For example, in the case of a virus, LTRs are required for genomic integration.

Lentirviral Vectors

The safety facet of suicide gene therapy relies on efficient delivery and stable, consistent expression of both the therapeutic and the cytotoxic effector genes. LVs transduce a wide range of dividing and non-dividing cell types with high efficiency, conferring stable, long-term expression of the transgene²⁵⁻²⁷.

The use of lentivirus-based gene transfer techniques relies on the in vitro production of recombinant lentiviral particles carrying a highly deleted viral genome in which the transgene of interest is accommodated. In particular, the recombinant lentivirus are recovered through the in trans coexpression in a permissive cell line of (1) the packaging constructs, i.e., a vector expressing the Gag-Pol precursors together with Rev (alternatively expressed in trans); (2) a vector expressing an envelope receptor, generally of an heterologous nature; and (3) the transfer vector, consisting in the viral cDNA deprived of all open reading frames, but maintaining the sequences required for replication, incapsidation, and expression, in which the sequences to be expressed are inserted.

In one embodiment the Lentigen lentiviral vector described in Lu, X. et al. Journal of gene medicine (2004) 6:963-973 is used to express the modified tmpk molecules.

In an embodiment the application comprises a lentiviral vector expressing a modified tmpk molecule fused to a detection cassette. In one embodiment the lentiviral vector comprises a 5′-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5′-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3′-splice site (SA), Elongation factor (EF) 1-alpha promoter and 3′-Self inactivating LTR(SIN-LTR). It will be readily apparent to one skilled in the art that optionally one or more of these regions is substituted with another region performing a similar function.

Gene therapy requires the transgene product to be expressed at sufficiently high levels. Enhancer elements can be used to increase expression of modified tmpk molecules or increase the lentiviral integration efficiency. In one embodiment the lentiviral vector further comprises a nef sequence. In a preferred embodiment the lentiviral further comprises a cPPT sequence which enhances vector integration. The cPPT acts as a second origin of the (+)-strand DNA synthesis and introduces a partial strand overlap in the middle of its native HIV genome. The introduction of the cPPT sequence in the transfer vector backbone strongly increased the nuclear transport and the total amount of genome integrated into the DNA of target cells. In an alternate preferred embodiment, the lentiviral vector further comprises a Woodchuck Posttranscriptional Regulatory Element (WPRE). The WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells. The addition of the WPRE to lentiviral vector results in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo. In a further preferred embodiment, the lentiviral vector comprises both a cPPT sequence and WPRE sequence.

The vector also comprises in an alternate embodiment an internal ribosome entry site (IRES) sequence that permits the expression of multiple polypeptides from a single promoter. For example the IRES can permit the expression of a therapeutic gene. For example, the therapeutic gene is in one embodiment, alpha-galactosidase A (NM_(—)000169). In another embodiment, the therapeutic gene is codon optimized alpha-galactaside A (COalphaGalA:all or part of SEQ ID NO:46; optionally the coding sequence of SEQ ID NO:46). In an embodiment, the COalphaGalA comprises all or part of SEQ ID NO:46, all of the coding sequence of SEQ ID NO:46, and/or a sequence with at least 95% identity to SEQ ID NO:46, wherein the sequence is not identical to the polynucleotide having accession number NM_(—)000169 and/or encodes SEQ ID NO:47, any of which wherein T is optionally substituted with U. Codon optimized alphaGalA refers to a alphaGalA coding sequence that is optimized for human codon usage.

In another embodiment the integrating vector is pHR′-cppt-EF-IRES-W-SIN. The IRES sequence is alternatively spliced out. For example where the construct comprises a tmpk/detection fusion, and no additional polypeptide, the IRES can be spliced out and/or the tmpk/detection fusion can be cloned in upstream such that the IRES is not utilized. In one embodiment the vector construct comprises pHR′-cppt-EF-tmpk/detection cassette-W-SIN. In another embodiment, the vector construct comprises pHR′-cppt-EF tmpk/detection cassette-IRES-W-SIN. In a more specific embodiment, the vector construct comprises pHR′-cppt-EF-CD19Δ/TmpkF105YR200A-W-SIN. In an alternate embodiment the vector construct comprises pHR′-cppt-E F-CD19Δ/TmpkF105YR200A-IRES-therapeutic gene-W-SIN. Additionally it will be readily apparent to one skilled in the art that optionally one or more of these elements can be added or substituted with other regions performing similar functions.

In addition to IRES sequences, other elements which permit expression of multiple polypeptides are useful. In one embodiment the vector comprises multiple promoters that permit expression more than one polypeptide. In another embodiment the vector comprises a protein cleavage site that allows expression of more than one polypeptide. Examples of protein cleavage sites that allow expression of more than one polypeptide comprise those listed in the following articles which are incorporated by reference: Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy. Klump H, Schiedlmeier B, Vogt B, Ryan M, Ostertag W, Baum C. Gene Ther. 200; 8(10):811-7; A picornaviral 2A-like sequence-based tricistronic vector allowing for high-level therapeutic gene expression coupled to a dual-reporter system Mark J. Osborn, Angela Panoskaltsis-Mortari, Ron T. McElmurry, Scott K. Bell, Dario A. A. Vignali, Martin D. Ryan, Andrew C. Wilber, R. Scott Mclvor, Jakub Tolar and Bruce R. Blazar. Molecular Therapy 2005; 12 (3), 569-574; Development of 2A peptide-based strategies in the design of multicistronic vectors. Szymczak A L, Vignali D A. Expert Opin Biol Ther. 2005; 5(5):627-38; Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Szymczak A L, Workman C J, Wang Y, Vignali K M, Dilioglou S, Vanin E F, Vignali D A. Nat. Biotechnol. 2004; 22(5):589-94. It will be readily apparent to one skilled in the art that other elements that permit expression of multiple polypeptides which identified in the future are useful and may be utilized in the vectors of the application.

Viral Regulatory Elements

The viral regulatory elements are components of vehicles used to introduce nucleic acid molecules into a host cell. The viral regulatory elements are optionally retroviral regulatory elements. For example, the viral regulatory elements may be the LTR and gag sequences from HSC1 or MSCV. The retroviral regulatory elements may be from lentiviruses or they may be heterologous sequences identified from other genomic regions.

One skilled in the art would also appreciate that as other viral regulatory elements are identified, these may be used with the nucleic acid molecules of the application.

Variations of Nucleic Acid Molecules Modifications

Many modifications may be made to the nucleic acid molecule DNA sequences including vector sequences disclosed in this application and these will be apparent to one skilled in the art. The application includes nucleotide modifications of the sequences disclosed in this application (or fragments thereof) that are capable of directing expression or being expressed in mammalian cells. Modifications include substitution, insertion or deletion of nucleotides or altering the relative positions or order of nucleotides.

Sequence Identity

The nucleic acid molecules of the application also include nucleic acid molecules (or a fragment thereof) having at least about: 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or, most preferred, at least 99% or 99.5% identity to a nucleic acid molecule of the application and which are capable of expression of nucleic acid molecules in mammalian cells. Identity refers to the similarity of two nucleotide sequences that are aligned so that the highest order match is obtained. Identity is calculated according to methods known in the art. For example, if a nucleotide sequence (called “Sequence A”) has 90% identity to a portion of SEQ ID NO: 11, then Sequence A will be identical to the referenced portion of SEQ ID NO: 11 except that Sequence A may include up to 10 point mutations (such as substitutions with other nucleotides) per each 100 nucleotides of the referenced portion of SEQ ID NO: 11.

Sequence identity (each construct in an embodiment without a coding nucleic acid molecule insert) is preferably set at least about: 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or, most preferred, at least 99% or 99.5% identity to the sequences provided in for example SEQ ID NO:13 and/or SEQ ID NO:14 and/or its complementary sequence. Sequence identity will preferably be calculated with the GCG program from Bioinformatics (University of Wisconsin). Other programs are also available to calculate sequence identity, such as the Clustal W program (preferably using default parameters; Thompson, J D et al., Nucleic Acid Res. 22:4673-4680).

Hybridization

The application includes DNA that has a sequence with sufficient identity to a nucleic acid molecule described in this application to hybridize under stringent hybridization conditions (hybridization techniques are well known in the art). The present application also includes nucleic acid molecules that hybridize to one or more of the sequences of for example SEQ ID NO:11 and/or SEQ ID NO:12 or its complementary sequence. Such nucleic acid molecules preferably hybridize under high stringency conditions (see Sambrook et al. Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). High stringency washes have preferably have low salt (preferably about 0.2% SSC) and a temperature of about 50-65° C. and are optionally conducted for about 15 minutes.

Prodrugs

A prodrug refers to a pharmacological substance (drug) which is administered in an inactive form (or significantly less active form, eg at least 90% or at least 95% less active than the active drug form). Once administered, the prodrug is metabolised in the body (in vivo) into the active compound and these metabolites provide cytotoxicity against the cells.

A prodrug is useful in combination with suicide gene therapy strategies. Suicide genes that make transduced cells susceptible to a molecule that is not ordinarily toxic function as a safety mechanism. The most commonly used suicide gene is the thymidine kinase gene from herpes simplex type I virus (HSV1-tk).

AZT is an example of a nucleoside prodrug that is poorly phosphorylated by thymidylate kinase enzymes. Other thymidine and uracil analogs are known and would be useful as prodrugs for killing cells expressing modified tmpk. Other known thymidine and uracil analogues that are useful comprise d4T and 5-FU. Additional thymidine and uracil analogs are known in the art. (J Med. Chem. 1996 39(17):3412-7 Synthesis and evaluation of novel thymidine analogs as antitumor and antiviral agents. Chen X, Bastow K, Goz B, Kucera L S, Morris-Natschke S L, Ishaq K S).

In a preferred embodiment, the prodrug administered is AZT. In an alternate embodiment the prodrug is a thymidine analog that is a substrate for modified tmpk enzymes. In another embodiment the prodrug is a uracil analog.

Prodrugs may require more than one enzyme activation. For example ganciclovir requires phosphorylation by thymidine kinase as well as a second phosphorylation guanylate kinase. In an embodiment of the present application, a method of tandem expression of modified tmpk and additional kinases required for prodrug is provided.

Tmpk/Detection Cassette Expressing Cells

The application also relates to a cell (for example, isolated cell in vitro, a cell in vivo, or a cell treated ex vivo and returned to an in vivo site) containing a nucleic acid molecule of the application.

The term “a cell” includes a single cell as well as a plurality or population of cells. Administering a composition to a cell includes both in vitro and in vivo administrations.

The term “a” as used herein means one or more than one.

Cells transfected with a nucleic acid molecule such as a DNA molecule, or transduced with the nucleic acid molecule such as a DNA or RNA virus vector construct, encoding a tmpk/detection cassette fusion are optionally used, for example, in bone marrow or cord blood cell transplants according to techniques known in the art. Examples of the use of transduced bone marrow or cord blood cells in transplants are for ex vivo gene therapy of Adenosine deaminase (ADA) deficiency. Other cells which are optionally transfected or transduced either ex vivo or in vivo include purified stem cells (of embryonic or later ontogeny), as described above.

Cells expressing tmpk/detection cassette optionally express a therapeutic gene, for example a therapeutic gene described herein.

Host Cells

The application also relates to a host cell (isolated cell in vitro, a cell in vivo, or a cell treated ex vivo and returned to an in vivo site) containing a nucleic acid molecule of the application.

The term “a cell” includes a single cell as well as a plurality or population of cells. Administering a composition to a cell includes both in vitro and in vivo administrations

Cells transfected with a nucleic acid molecule such as a DNA molecule, or transduced with the nucleic acid molecule such as a DNA or RNA virus vector construct, are optionally used, for example, in bone marrow or cord blood cell transplants according to techniques known in the art. Examples of the use of transduced bone marrow or cord blood cells in transplants are for ex vivo gene therapy of Adenosine deaminase (ADA) deficiency. Other cells which are optionally transfected or transduced either ex vivo or in vivo include purified stem cells (of embryonic or later ontogeny), as described above.

In certain embodiments, the cell is a cancer cell.

The term “cancer cell” includes cancer or tumor-forming cells, transformed cells or a cell that is susceptible to becoming a cancer or tumor-forming cell.

Cell Types for Transplant

Modified tmpk/detection cassette fusion molecules are usefully introduced into any cell type ex vivo where it is desirable to provide a mechanism for killing the modified tmpk/detection cassette fusion expressing cells. Cell types that are useful in one embodiment of the present application include, but are not limited to, stem cells (both embryonic and of later ontogeny as well as mesenchymal stem cells, induced pluripotent stem cells (IPS)), cord blood cells, and immune cells such as T cells, bone marrow cells and peripheral blood mononuclear cells. T-cells are optionally CD4 positive, CD8 positive or double positive. In addition, T cells are optionally mature T cells. In one embodiment T cells are transduced with modified tmpk/detection cassette fusion molecules, isolated and transplanted in a host. In another embodiment the T cells are mature T cells. In an alternate embodiment stem cells are transduced, isolated and transplanted in a host. In a further embodiment, IPS cells are transduced, isolated and transplanted in a host. In yet a further embodiment, mesenchymal stem cells are transduced isolated and transplanted in a host.

Cell lines are optionally transduced. For example human T cell leukemia Jurkat T cells, human erythro-leukemic K562 cells, human prostate cell lines DU145 and PC3 cells are optionally transduced or transfected with modified tmpk/detection cassette fusion molecules.

Tissue Specific Expression

In an alternate embodiment of the application, the modified tmpk/detection cassette fusion expressing cells express tmpk/detection cassette fusion under the control of a tissue or cell specific promoter providing expression in a tissue specific manner. Expression of modified tmpk/detection cassette fusion molecules is optionally targeted to tumor cells using promoters that are active in tumor cells.

Accordingly, in one aspect of the application, vector constructs comprising modified tmpk/detection cassette fusion molecules are provided that result in tissue or cell specific expression of the modified tmpk/detection cassette fusion molecules. Tissue and cell specific expression of modified tmpk/detection cassette fusion is typically accomplished using promoters operably linked with the modified tmpk/detection cassette fusion, which limit expression of modified tmpk/detection cassette fusion to cells or tissues. One skilled in the art will recognize that a variety of promoter sequences that direct tissue or cell specific expression are useful to direct tissue or cell specific expression of modified tmpk/detection cassette fusion. For example, one skilled in the art will readily recognize that liver specific expression is accomplished using a liver specific promoter. Modified tmpk/detection cassette fusion expression is readily limited to a variety of cell and tissue types. Examples include, but are not limited to, liver, pancreas and T cells. Examples of liver specific promoters include, but are not limited to, the transthyretin promoter, albumin promoter, alpha feto protein promoter. Examples of other cell specific promoters include, but are not limited to, islet cell specific promoters such as the insulin promoter, and T cell specific promoters such as CD4-promoter. In another embodiment, expression of modified tmpk/detection cassette fusion is inducible. The hypoxia-inducible promoter is optionally used to direct expression of a cytoprotective gene such as but not limited to erythropoietin. Introduction of a cytoprotective gene under the control of an inducible promoter such as the hypoxia inducible promoter is useful, to prevent the severe tissue damage by hypoxia.

If the transduced cells cause some problems or if it is otherwise desirable to clear the transduced cells, the transduced cells are optionally cleared (killed) by suicide effect by administering prodrug to the transduced cells or subject.

Tumor cell specific expression is accomplished using a tumor specific promoter. Tumor specific promoters comprise the progression elevated gene-3 (PEG-3) promoter. This promoter functions selectively in divergence cancer cells with limited activity in normal cells, for tumor cell-specific expression. The transduced tumor cells are specifically killed by the prodrug.

Methods of Isolation

In one aspect of the present application, methods for expressing a modified tmpk/detection cassette fusion molecule in cells for transplant are provided. After transduction or transfection with vector constructs comprising modified tmpk detection cassette fusion molecules or control molecules, cells expressing these molecules are optionally isolated by a variety of means known in the art. As the detection cassette is fused to tmpk, detection of cells expressing the detection cassette protein or nucleic acid identify cells expressing tmpk. Accordingly the molecule encoded by the detection cassette is used to isolate modified tmpk/detection cassette fusion positive cells. In certain embodiments, the cells are isolated by cell sorting or flow cytometry using an antibody to the detection cassette encoded molecule. Additionally cell sorting is useful to isolate modified tmpk/detection cassette fusion expressing cells where the detection cassette is a fluorescent protein such as EGFP. Cells expressing modified tmpk/detection cassette fusion or control molecules are, in an alternate embodiment, isolated using magnetic sorting. Additionally, cells may be isolated by drug selection. In one embodiment, a vector comprising a drug resistance gene and a modified tmpk/detection cassette fusion molecule is introduced into cells. Examples of drug resistance genes include, but are not limited to, neomycin resistance gene, blasticidin resistance gene (Bsr), hygromycin resistance gene (Hph), puromycin resistance gene (Pac), Zeocin resistance gene (Sh ble), FHT, bleomycin resistance gene and ampicillin resistance gene After transduction or transfection, cells expressing modified tmpk/detection cassette fusion or control molecules and the drug resistance gene are selected by adding the drug that is inactivated by the drug resistance gene. Cells expressing the drug resistance gene survive while non-transfected or non-transduced cells are killed. A person skilled in the art would be familiar with the methods and reagents required to isolate cells expressing modified tmpk/detection cassette fusion molecules.

Methods of and Uses for Treatment Treatment of Cancer

The present application provides modified tmpk/detection cassette fusion molecules that are useful for the reduction of cell proliferation, for example for treatment of cancer. The present application also provides methods of expressing modified tmpk/detection cassette fusion molecules for the reduction of cell proliferation, for example for treatment of cancer.

Modified tmpk/detection cassette fusion is introduced into cells that are used for transplant or introduced directly in vivo in mammals, preferably a human. The modified tmpk/detection cassette fusion molecules are typically introduced into cells ex vivo (e.g. in vitro) using methods known in the art. Methods for introducing tmpk/detection cassette fusion molecules comprise transfection, infection, electroporation. These methods optionally employ liposomes or liposome like compounds.

In one embodiment, modified tmpk/detection cassette fusion molecules are used to treat cancer by adoptive therapy. Adoptive therapy or adoptive (immuno)therapy refers to the passive transfer of immunologically competent tumor-reactive cells into the tumor-bearing host to, directly or indirectly, mediate tumor regression. The feasibility of adoptive (immuno)therapy of cancer is based on two fundamental observations. The first of these observations is that tumor cells express unique antigens that can elicit an immune response within the syngeneic (genetically identical or similar especially with respect to antigens or immunological reactions) host. The other is that the immune rejection of established tumors can be mediated by the adoptive transfer of appropriately sensitized lymphoid cells. Clinical applications include transfer of peripheral blood stem cells following non-myeloablative chemotherapy with or without radiation in patients with lymphomas, leukemias, and solid tumors.

In one aspect of the present application, donor T cells or stem cells (either embryonic or of later ontogeny) are transduced with vectors comprising modified tmpk/detection cassette fusion molecules. Cells expressing said modified tmpk/detection cassette fusion are isolated and adoptively transferred to a host in need of treatment. In one embodiment the bone marrow of the recipient is T-cell depleted. Methods of adoptive T-cell transfer are known in the art (J Translational Medicine, 2005 3(17): doi; 0.1186/1479-5876-3-17, Adoptive T cell therapy: Addressing challenges in cancer immunotherapy. Cassian Yee). This method is used to treat solid tumors and does not require targeting the modified tmpk/detection cassette fusion expressing T-cells to the tumor since the modified tmpk/detection cassette fusion donor T-cells will recognize the different MHC class molecules present in the recipient host resulting in cytotoxic killing of tumor cells.

Another aspect of the application provides for the treatment of solid tumors by injecting vectors carrying modified tmpk/detection cassette fusion molecules directly into the tumor. Methods of introducing modified tmpk/detection cassette fusion directly in vivo in a mammal, preferably a human, comprise direct viral delivery, microinjection, in vivo electroporation, and liposome mediated methods.

Thymidine kinase has been introduced by injection directly into the site of a tumor to examine results of the technique as a cancer therapeutic treatment (Chevez-Barrios P, Chintagumpala M, Mieler W, Paysse E, Boniuk M, Kozinetz C, Hurwitz M Y, Hurwitz R L. Response of retinoblastoma with vitreous tumor seeding to adenovirus-mediated delivery of thymidine kinase followed by ganciclovir. J Clin Oncol. 2005 Nov. 1; 23(31):7927-35. Sterman D H, Treat J, Litzky L A, Amin K M, Coonrod L, Molnar-Kimber K, Recio A, Knox L, Wilson J M, Albelda S M, Kaiser L R. Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma. Hum Gene Ther. 1998 May 1; 9(7):1083-92). The tmpk/detection cassette fusion molecules of the present application are optionally introduced directly into the site of a tumor to reduce proliferation of tumor cells, for example, to treat cancer.

In one embodiment, cells are transfected or transduced ex vivo with modified tmpk/detection cassette fusion vector constructs. In an optional embodiment, the vector construct comprises a lentiviral vector.

Graft Versus Leukemia

In addition, the application provides, in one aspect, a method of treating leukemia. Donor T cells or stem cells are transduced with vectors comprising modified tmpk/detection cassette fusion molecules, cells expressing said modified tmpk/detection cassette fusion are isolated and transplanted to a host in need of treatment. The transplanted cells induce a graft versus leukemia effect. If the transplanted cells induce graft versus host disease, the transplanted cells can be killed by administering a prodrug.

Graft versus leukemia refers to using donor transplant cells to kill host leukemic cells. Introduced cells will often also attack the cancer cells that still may be present after transplant. This was first documented in acute leukemia, and this phenomenon has been called “graft-versus-leukemia” effect. Similar effects have been observed in malignant lymphoma, myeloma, and perhaps even some solid tumors. For certain diseases, such as chronic myelogenous leukemia (CML), the graft-versus-leukemia (GvL) effect may well be the most important reason that allogeneic transplants are successful in curing the disease.

Method of Treating Graft Versus Host Disease (GVHD)

Graft versus host disease is a common complication of allogeneic bone marrow transplantation (BMT). After bone marrow transplantation, T cells present in the graft, either as contaminants or intentionally introduced into the host, attack the tissues of the transplant recipient. Graft-versus-host disease can occur even when HLA-identical siblings are the donors. HLA-identical siblings or HLA-identical unrelated donors (called a minor mismatch as opposed to differences in the HLA antigens, which constitute a major mismatch) often still have genetically different proteins that can be presented on the MHC.

Graft versus host disease is a serious complication of transplant and can lead to death in patients that develop severe graft versus host disease (the clinical manifestations of graft versus host disease are reviewed in Socie G. Chronic graft-versus-host disease: clinical features and grading systems. Int J. Hematol. 2004 April; 79(3):216-20). Viral thymidine kinase has been introduced into transplant cells and used in combination with drugs such as ganciclovir to determine the results in individuals who develop graft versus host disease. (Bonini C, Ferrari G, Verzeletti S, Servida P, Zappone E, Ruggieri L, Ponzoni M, Rossini S, Mavilio F, Traversari C, Bordignon C HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science. 1997 June. 13; 276(5319):1719-24; Bondanza A, Valtolina V, Magnani Z, Ponzoni M, Fleischhauer K, Bonyhadi M, Traversari C, Sanvito F, Toma S, Radrizzani M, La Seta-Catamancio S, Ciceri F, Bordignon C, Bonini C Suicide gene therapy of graft-versus-host disease induced by central memory human T lymphocytes. Blood. 2005.)

While donor T-cells are undesirable as effector cells of graft-versus-host-disease, they are valuable for engraftment by preventing the recipient's residual immune system from rejecting the bone marrow graft (host-versus-graft). Additionally, as bone marrow transplantation is frequently used to cure malignant disorders (most prominently the leukemias), donor T-cells have proven to have a valuable graft-versus-tumor (GVT, graft versus leukemia described above) effect. A great deal of current research on allogeneic bone marrow transplantation involves attempts to separate the undesirable graft-vs-host-disease aspects of T-cell physiology from the desirable graft-versus-tumor effect.

The present application provides, in one embodiment, methods of treating transplant patients that develop graft versus host disease by administering compounds of the application (eg. tmpk/detection cassette fusion molecules, constructs or compositions used in combination with drugs) to a mammal in need thereof. In another embodiment, the application provides a method of promoting graft versus tumor effect by administering compounds of the application to a mammal in need thereof.

Treating Inherited Genetic Diseases and Safety Component for Gene Therapy

One problem with the use of gene therapy to stably introduce exogenous polynucleotides is the potential to develop a gene therapy related disease such as cancer. A gene therapy vector can integrate into a DNA region that could cause cancer in the gene therapy patient.

In one embodiment of the application, tmpk/detection cassette fusion moleucules are useful as a safety component in gene therapy constructs. It is clear to one skilled in the art that the tmpk/detection cassette fusion mutants are useful in combination with different therapeutic polynucleotides designed to treat a variety of conditions. The tmpk/detection cassette fusionsare useful in combination with a therapeutic polynucleotide that encodes a polypeptide that compensates for a deficient gene product. Examples of diseases that comprise a deficient gene product include, but not are limited to, Factor IX deficiency, Factor VIII deficiency, Gaucher disease, SCID, MPS, cystic fibrosis, Fabry disease, Farber disease, sickle cell disease, chronic granulomatous disorder (CGD). In this aspect of the application, vectors comprising a tmpk/detection cassette fusion and a deficient gene are introduced into cells ex vivo such as bone marrow cells or provided systemically to a patient deficient in the gene product. Systemically introduced vectors can integrate into host cells forming gene-modified cells. If the gene-modified cells cause a gene therapy related disease in the recipient model, a prodrug is administered to the recipient that kills the gene-modified cells.

Isolated Virus

Another aspect relates an isolated virus comprising a viral genome that encodes:

-   -   a modified mammalian thymidylate kinase (tmpk) polynucleotide         wherein the modified mammalian tmpk polynucleotide encodes a         modified mammalian tmpk polypeptide that increases         phosphorylation of a prodrug relative to phosophorylation of the         prodrug by wild type mammalian tmpk polypeptide;     -   a detection cassette polynucleotide fused to the tmpk         polynucleotide;         wherein the isolated virus is infective for a mammalian cell. A         person skilled in the art would readily recognize that a number         of minimal elements are necessary for infection as described         herein. For example flanking LTR sequences are necessary in         retroviruses for genomic integraton, a promoter operabley linked         to the insert polynucleotide eg. the tmpk/detection cassette         fusion.

A person skilled in the art would readily understand that an RNA virus comprises a RNA genome such that corresponding RNA components and sequences of the components and sequences described herein would be found in an RNA virus genome. Accordingly sequences such as tmpk/detection cassette fusion sequences described herein also include sequences wherein T is optionally substituted with U.

Methods of making and isolating virus are described herein, for example in the Examples and further are known in the art.

The isolated virus can be a retrovirus, optionally a lentivirus or an adenovirus. Other viruses described herein are also contemplated. In an embodiment, the isolated virus is a clinical grade virus.

The modified tmpk and the detection cassette are optionally any modified tmpk and dectection cassettes described herein.

In an embodiment, the isolated virus further comprises a therapeutic cassette, optionally any therapeutic cassette described herein, including adenosine deaminase, γc interleukin receptor subunit, α-galactosidase A, codon optimized α-galactosidase A, acid ceramidase, galactocerebrosidase, and CFTR molecules.

The isolated virus optionally infects a mammalian cell, optionally wherein the mammalian cell is selected from the group consisting of a stem cell, an embryonic stem cell, a mesenchymal stem cell, an induced pluripotent stem (IPS) cell, a hematopoietic cell, a T cell and a human cell.

The application also provides uses for treating cancer, inherited genetic diseases and cancer.

Further the application provides compositions, vector constructs the actuable destruction component, isolated viruses or cells expressing tmpk/detection cassette fusions for use as a pharmaceutical.

Also provides compositions, vector constructs the actuable destruction component, isolated viruses or cells expressing tmpk/detection cassette fusions, for treating cancer, for treating GVHD, or for treating an inherited genetic disorder (e.g gene therapy).

Furthermore, use of compositions, vector constructs the actuable destruction component, isolated viruses or cells expressing tmpk/detection cassette fusions for the manufacture of a medicament for treating cancer, for treating GVHD, or for treating an inherited genetic disorder (e.g gene therapy) is also provided.

Polynucleotides of Interest/Therapeutic Nucleic Acid Molecules

Cells transfected or transduced in vitro can be used for ex vivo gene therapy or as a research tool or for protein production. The nucleic acid molecules are also useful for gene therapy by transfecting or transducing cells in vivo to express a therapeutic polynucleotide/protein in addition to modified tmpk/detection cassette fusion. The therapeutic polynucleotide is alternatively referred to herein as the therapeutic cassette and/or therapeutic expression cassette. For example, if one were to upregulate the expression of a gene, one could insert the sense sequence into the nucleic acid molecule. If one were to downregulate the expression of the gene, one could insert the antisense sequence into the therapeutic expression cassette. Techniques for inserting sense and antisense sequences (or fragments of these sequences) would be apparent to those skilled in the art. The therapeutic nucleic acid molecule or nucleic acid molecule fragment may be either isolated from a native source (in sense or antisense orientations) or synthesized. It may also be a mutated native or synthetic sequence or a combination of these.

Examples of therapeutic coding nucleic acid molecules to be expressed include adenosine deaminase (ADA), γc interleukin receptor subunit, α-galactosidase A, codon optimized α-galactosidase A acid ceramidase, galactocerebrosidase, and transmembrane conductance regulator (CFTR) molecules.

Drug Discovery Platform

The present application also provides assays for identifying novel thymidine and uracil analog compounds that are useful as prodrugs in combination with modified tmpk/detection cassette fusion molecules. The thymidine analogs can be synthesized according to methods known in the art (J Med. Chem. 1996 39(17):3412-7 Synthesis and evaluation of novel thymidine analogs as antitumor and antiviral agents. Chen X, Bastow K, Goz B, Kucera L S, Morris-Natschke S L, Ishaq K S) and tested for the use as substrates by modified tmpk/detection cassette fusion polypeptides. Alternatively libraries of thymidine or uracil analogs can be synthesized and screened for compounds that can act as substrates for modified tmpk/detection cassette fusion polypeptides. Methods for the synthesis of molecular libraries are known in the art (Novel nucleotide analogues as potential substrates for TMPK, a key enzyme in the metabolism of AZT. Muller H C, Meier C, Balzarini J, Reinstein J. Nucleosides Nucleotides Nucleic Acids. 2003; 22(5-8):821-3).

In one aspect of the present application, compounds are identified using rational drug design and tested for their use as substrates for modified tmpk/detection cassette fusion polypeptides.

In one embodiment the assay comprises, a cell-based assay comprising the steps of:

-   -   i) introducing a modified tmpk/detection cassette fusion         molecule into a cell;     -   ii) providing a thymidine analog to the cell; and     -   iii) determining whether said thymidine analog is a substrate         for said modified tmpk/detection cassette fusion.

In an alternate embodiment, the assay comprises a cell free assay comprising the steps of:

-   -   i) providing an enzymatically active modified tmpk/detection         cassette fusion,     -   ii) providing a thymidine analog to the modified tmpk/detection         cassette fusion;     -   iii) determining whether said thymidine analog is a substrate         for said modified tmpk/detection cassette fusion.

The enzymatically active modified tmpk/detection cassette fusion can comprise an additional fusion such as a GST fusion protein. In one embodiment the assay is conducted in a test tube. In an alternative embodiment the assay is conducted in a micro-titer plate. The enzymatically active modified tmpk/detection cassette fusion can be free in solution or bound to beads such as sepharose beads. The determination of whether said thymidine analog is a substrate for said modified tmpk/detection cassette fusion can comprise the use radioactive phosphorus or non-radioactive means. The methods of assessing kinase activity and substrate utilization are well known in the art.

Pharmaceutical Compositions

The pharmaceutical compositions of this application used to treat patients having diseases, disorders or abnormal physical states could include an acceptable carrier, auxiliary or excipient.

The pharmaceutical compositions are optionally administered by ex vivo and in vivo methods such as electroporation, DNA microinjection, liposome DNA delivery, and virus vectors that have RNA or DNA genomes including retrovirus vectors, lentivirus vectors, Adenovirus vectors and Adeno-associated virus (AAV) vectors, Semliki Forest Virus. Derivatives or hybrids of these vectors are also useful.

Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration. The expression cassettes are optionally introduced into the cells or their precursors using ex vivo or in vivo delivery vehicles such as liposomes or DNA or RNA virus vectors. They are also optionally introduced into these cells using physical techniques such as microinjection or chemical methods such as coprecipitation.

The pharmaceutical compositions are typically prepared by known methods for the preparation of pharmaceutically acceptable compositions which are administered to patients, and such that an effective quantity of the nucleic acid molecule is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA).

On this basis, the pharmaceutical compositions could include an active compound or substance, such as a nucleic acid molecule, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and isoosmotic with the physiological fluids. The methods of combining the expression cassettes with the vehicles or combining them with diluents is well known to those skilled in the art. The composition could include a targeting agent for the transport of the active compound to specified sites within cells.

Method of Medical Treatment of Disease

Vectors containing the nucleic acid molecules of the application are typically administered to mammals, preferably humans, in gene therapy using techniques described below. The polypeptides produced from the nucleic acid molecules are also optionally administered to mammals, preferably humans. The application relates to a method of medical treatment of a mammal in need thereof, preferably a human, by administering to the mammal a vector of the application or a cell containing a vector of the application. A recipient, preferably human, who develops an adverse event, such as graft versus host disease, is typically administered a drug, such as AZT, that is a substrate for the modified tmpk molecules of the application. Diseases, such as blood diseases or neural diseases (neurodegenerative), that are readily treated are described in this application and known in the art (eg. diseases, such as thalassemia or sickle cell anemia that are treated by administering a globin gene as described in Canadian patent application no. 2,246,005). Blood diseases treatable by stem cell transplant include leukemias, myelodysplastic syndromes, stem cell disorders, myeloproliferative disorders, lymphoproliferative disorders phagocyte disorders, inherited metabolic disorders, histiocytic disorders, inherited erythrocyte abnormalities, inherited immune system disorders, inherited platelet abnormalities, plasma cell disorders, malignancies (See also, Medical Professional's Guide to Unrelated Donor Stem Cell Transplants, 4th Edition). Stem cell nerve diseases to be treated by neural stem cell transplantation include diseases resulting in neural cell damage or loss, eg. paralysis, Parkinson's disease, Alzheimer's disease, ALS, multiple sclerosis). The vector of the application is useful as a stem cell marker and to express genes that cause stem cells to differentiate (e.g. growth factor).

Gene Therapy

The application includes compositions and methods for providing a coding nucleic acid molecule to a subject such that expression of the molecule in the cells provides the biological activity of the polypeptide encoded by the coding nucleic acid molecule to those cells. A coding nucleic acid as used herein means a nucleic acid that comprises nucleotides which specify the amino acid sequence, or a portion thereof, of the corresponding protein. A coding sequence may comprise a start codon and/or a termination sequence.

The application includes methods and compositions for providing a coding nucleic acid molecule to the cells of an individual such that expression of the coding nucleic acid molecule in the cells provides the biological activity or phenotype of the polypeptide encoded by the coding nucleic acid molecule. The method also relates to a method for providing an individual having a disease, disorder or abnormal physical state with a biologically active polypeptide by administering a nucleic acid molecule of the present application. The method may be performed ex vivo or in vivo. Gene therapy methods and compositions are demonstrated, for example, in U.S. Pat. Nos. 5,869,040, 5,639,642, 5,928,214, 5,911,983, 5,830,880, 5,910,488, 5,854,019, 5,672,344, 5,645,829, 5,741,486, 5,656,465, 5,547,932, 5,529,774, 5,436,146, 5,399,346 and 5,670,488, 5,240,846. The amount of polypeptide will vary with the subject's needs. The optimal dosage of vector may be readily determined using empirical techniques, for example by escalating doses (see U.S. Pat. No. 5,910,488 for an example of escalating doses).

Various approaches to gene therapy may be used. The application includes a process for providing a human with a therapeutic polypeptide including: introducing human cells into a human, said human cells having been treated in vitro or ex vivo to insert therein a vector of the application, the human cells expressing in vivo in said human a therapeutically effective amount of said therapeutic polypeptide.

The method also relates to a method for producing a stock of recombinant virus by producing virus suitable for gene therapy comprising modified DNA encoding globin. This method preferably involves transfecting cells permissive for virus replication (the virus containing modified globin) and collecting the virus produced.

Cotransfection (DNA and marker on separate molecules) may be employed (see eg U.S. Pat. No. 5,928,914 and U.S. Pat. No. 5,817,492). As well, a detection cassette or marker (such as Green Fluorescent Protein marker or a derivative, CD19 or CD25) may be used within the vector itself (preferably a viral vector).

Polypeptide Production and Research Tools

A cell line (either an immortalized cell culture or a stem cell culture) transfected or transduced with a nucleic acid molecule of the application (or variants) is useful as a research tool to measure levels of expression of the coding nucleic acid molecule and the activity of the polypeptide encoded by the coding nucleic acid molecule.

The nucleic acid molecules are useful in research to deliver marker genes or antisense RNA to cells.

The application includes a method for producing a recombinant host cell capable of expressing a nucleic acid molecule of the application comprising introducing into the host cell a vector of the application.

The application also includes a method for expressing a polypeptide in a host cell of the application including culturing the host cell under conditions suitable for coding nucleic acid molecule expression. The method typically provides the phenotype of the polypeptide to the cell.

In these methods, the host cell is optionally a stem cell or a T cell.

Another aspect of the application is an isolated polypeptide produced from a nucleic acid molecule or vector of the application according to a method of the application.

EXAMPLES

The following non-limiting examples are illustrative of the present application:

Example 1 Materials and Methods cDNA Cloning of Human CD19 and Construction of Shuttle Vector

Full-length human CD19 (hCD19) cDNA was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) from the human Burkitt's lymphoma cell line (Raji) using primers CD19 F1 and CD19 R1 described below. The cloned PCR product was directly ligated into the TA-vector, pPCR-script SK(+)(Stratagene) to give pPCR-CD19full. A truncated form of hCD19 (CD19D), which has only the extracellular and transmembrane domains, but lacks the cytoplasmic domain, was generated by inverse-PCR from pPCR-script SK(+)-CD19 using primers CD19 F2 and CD19 R2 described below, to give pPCR-CD19D. Following the sequence confirmation of cDNA inserts in pPCR-script SK(+)-CD19D, the cloned cDNA fragments were then subcloned into the EcoRI site of the shuttle vector pSV-IRES to give pSV-IRES-CD19D. The primer sequences used for cloning of human CD19 cDNA as follows: CD19 F1: 5′-atgccacctcctcgcctcctcttcttcc-3′ (SEQ ID NO: 23) and CD19 R1: 5′-tcacctggtgctccaggtgccc-3′ (SEQ ID NO: 24). The truncated construct was made by inverse-PCR using primers CD19 F2: 5′-ccgccaccgcggtggagctccag-3′ (SEQ ID NO: 25) and CD19 R2: 5′-ttaaagatgaagaatgcccacaaggg-3′ (SEQ ID NO: 26).

cDNA Cloning of Human Thymidylate Kinase, Construction of Bicistoronic Lentiviral Expression Vectors and Preparation of High-Titer Virus

To clone wild-type human thymidylate monophosphate kinase (tmpk) cDNA, peripheral blood mononuclear cells (PBMNCs) were isolated from heparinized blood obtained from healthy human donors by Ficoll-Hypaque (Amersham-Pharmacia) separations. Wild-type human tmpk cDNA was amplified by PCR using first strand complementary DNA (cDNA) generated by reverse-transcription from total RNA extracted from the PBMNCs using TRIZOL reagent (Invitrogen). PCR products for wild-type tmpk and each modified version of human tmpk cDNA, such as R200A, F105Y, and R16GLL, which was constructed by Dr. A. Lavie at the University of Illinois at Chicago, were subcloned into pPCR-scriptSK(+) and sequenced. Following the sequence confirmation, each cDNA was first subcloned into shuttle vector pSV-IRES-CD19D to construct a bicistronic cassette consisting the suicide gene, internal ribosomal entry site (IRES) derived from encephalomyocardiTUS virus (EMCV), and the truncated form of human CD19. This bicistronic expression cassette with tmpk and hCD19, flanked by an EMCV IRES. Then subcloned into HIV-1 based recombinant lentiviral plasmid vector used in the production of lentivirus, pHR′-cPPT-EF-W-SIN (pHR′). The expression of gene of interests was controlled by the internal EF1a promoter. As a control for the transduction experiments, the inventors used both pHR′-cPPT-EF-IRES-hCD19-W-SIN and pHR′-cPPT-EF-EGFP-W-SIN vectors carrying either IRES-hCD19 or the enhanced GFP (EGFP) cDNAs, respectively.

VSVG-pseudotyped LVs, including an EGFP marking vector (LV-EGFP), were generated by transient transfection of 293T cells (kindly provided by Dr. Robert Pawliuk, Division of Health Sciences and Technology, MIT, Cambridge, Mass.) using the three-plasmid system (the aforementioned LV plasmid constructs, the packaging plasmid pCMVΔR8.91, and the VSVG envelope encoding plasmid pMD.G). The transfections were performed with either FuGENE6 (Roche Applied Science, Indianapolis, Ind.) or CaPO4-precipitation methods. Viral supernatants were harvested 48 h later and concentrated by ultracentrifugation at 50,000×g for 2 h at 4° C. The concentrated viral supernatants were serially diluted and titered on 293T cells (ATCC, Manassas, Va.). Table 1 lists the titers of virus used in these experiments. Flow cytometric analyses were performed 72 h later using a FACSCalibur (BD Biosciences, San Jose, Calif.) for evaluating EGFP or hCD19 expression after staining with monoclonal PE-conjugated mouse anti-human CD19. Titers are expressed as infectious particles (IP)/mL.

TABLE 1 Titer of LVs on 293T cells used in this study Transgene Detection Titer (IP/mL) EGFP EGFP 1.4 × 10⁸ Tmpk (wild-type)-IRES-hCD19 CD19 2.3 × 10⁸ Tmpk (R200A)-IRES-hCD19 CD19 3.5 × 10⁸ Tmpk (F105Y + R200A)-IRES-hCD19 CD19 5.9 × 10⁸ Tmpk (R16G Large Lid)-IRES-hCD19 CD19 1.5 × 10⁹ IRES-hCD19 CD19 1.4 × 10⁹

Transduction and Analysis of Transgene Expression by Flow Cytometric Analysis.

Human T lymphoma cell line, Jurkat, and human erythro-leukemic cell line, K562, were maintained in RPMI 1640 supplemented with 10% FBS, 100 U/ml of penicillin, and streptomycin to 100 μg/ml. Cells were infected with concentrated virus stocks using an MOI of 10 in the presence of 8 μg/ml protamine sulfate. Infected cells were then kept in culture for 5 days prior to evaluating gene transfer efficiency. Gene transfer efficiencies were measured by flow cytometry using a monoclonal anti-human CD19-antibody conjugated with phycoerythrin (PE). About 10⁶ non-transduced and virally transduced cells were incubated for 15 min with the antibody or the corresponding IgG₁ isotype control antibody at 4° C. Cells were washed with phospahate-buffered saline (PBS). Cell analysis was performed on a FACS Calibur and data were analyzed using Cell Quest software. Single-cell clones were obtained by limiting dilution and clones with the highest expression of CD19 were selected.

Western Blot Analysis of Tmpk-Overexpression by LV-Transduction in Jurkat Cells.

Tmpk overexpression in the infected cells were examined by Western blot analysis using rabbit anti-human tmpk antibody (gift from Dr. Manfred Konrad, Maxplank Institute) as well as mouse anti-human beta-actin as an internal control for the blot. Total cell lysates were resolved by 12% SDS-polyacrylamide gels (SDS-PAGE) and transferred onto polyvinylidene difluoride filters (Millipore, Billerica, Mass.). Filters were blocked with 5% fat free skim milk in Tris-buffered saline (TBS) with 0.05% Tween 20 (TBST) for 1 hr at room temperature. Human tmpk overexpression was elucidated using rabbit anti-human tmpk antiserum, diluted to 1 in 5000. Protein loading amounts in each well was confirmed with an anti-beta actin antibody diluted 1:5000. Blots were probed with a secondary anti-rabbit IgG (diluted 1:5000) or anti-mouse IgG (diluted 1:5000) horseradish peroxidase-conjugated antibodies, and protein bands were detected using an enhanced chemiluminesence kit (Perkin Elmer, Norwalk, Conn.) and Kodak BioMAX XAR film.

Comparison of Transduction Efficiencies and hCD19 Expression Levels in LV-Transduced Jurkat Cells.

Cells were infected with concentrated virus stocks using an MOI of 10 in the presence of 8 μg/ml protamine sulfate. Infected cells were then kept in culture for 5 days prior to evaluating gene transfer efficiency. Gene transfer efficiencies were measured by flow cytometry using a monoclonal anti-human CD19-antibody conjugated to phycoerythrin (PE). About 10⁶ non-transduced and virally transduced cells were incubated for 15 min with the antibody or the corresponding IgG₁ isotype control antibody at 4° C. Cells were washed with phospahate-buffered saline (PBS). Cell analysis was performed on a FACS Calibur and data were analyzed using Cell Quest software. Single-cell clones were obtained by limiting dilution, and clones with the highest expression of CD19 were selected. Percentages indicate EGFP or CD19 expression and mean fluorescence intensity (MFI) values indicate the levels of expression levels in the cells.

Determination of AZT-Sensitivity of Jurkat (Human T Cell Line) Transduced with LV-tmpk-IRES-hCD19 and Mutant Forms.

Transduced Jurkat cells and the single-cell clones were seeded in 96 well plates (2×10⁵/well) in 200 μl of medium containing increasing concentrations of AZT (0, 1, 10 and 100 μM). The medium was changed daily. After 4 days of culture, cell viability was determined by MTT assay (Promega). **, P<0.01, n=3. Data are expressed as mean±standard error of mean (SEM).

Induction of Apoptosis by Addition of 100 μM AZT in LV-tmpk-Transduced Jurkat Cells.

Cells were seeded in 24 well plates (10⁶/well) in 1 ml of medium with or without 100 μM of AZT. The medium was changed daily. After 4 days of culture, induction of apoptosis in the cells were analyzed by annexin-V staining according to the manufacturer's protocol (Annexin V-APC: BD Pharmingen). **, P<0.01, n=3. Data are expressed as mean±SEM.

Determination of AZT-Metabolites in the Cells Treated with 100 μM AZT.

The cells were cultured in the presence of 100 μM AZT for 36 hrs. 10⁷ cells were homogenized by sonication in 100 μl of 5% (w/v) trichloroacetic acid. The supernatant is collected after homogenate had been centrifuged at 10,000×g for 15 min at 4° C. The trichloroacetic acid was removed by extraction with an equal volume of 20% tri-n-octylamine in pentane. The neutralized aqueous fraction is directly injected into HPLC. Separation of AZT and its metabolites was performed on a C18 column (Waters) with a mobile phase composed of 0.2 M phosphate buffer containing 4 mM tetrabutylammonium hydrogen sulfate (pH 7.5) and acetonitrile in the ratio of 97:3 (v/v). The mobile phase was pumped at a flow rate of 1.5 ml/min. The UV absorbance was monitored at 270 nm. Five million cell equivalents were injected and analyzed in triplicate.

AZT-Mediated Loss of Mitochondrial Function is Induced by Expression of TMPK-LargeLid.

Cells (10⁶ cells) treated with (shown (+) in figure) or without (−) 100 μM AZT were stained with JC-1 for 15 min at 37° C., and then were analyzed by flow cytometry. ***, P<0.001, n=3.

Cellular Proliferation is not Always a Prerequisite for AZT-Induced Apoptosis.

Cells were seeded in 24-well plates (10⁶/well) in 1 ml of medium containing 0 (shown in AZT (−) in figure) or 100 μM of AZT (shown in AZT (+)) with or without 5 μM indirubin-3′-oxime (Figure (B) and (A), respectively). The medium was refreshed daily. After 2 days of culture, induction of apoptosis by AZT was analyzed by annexin V staining according to the manufacturer's protocol described. **, P<0.01, n=3. Data are expressed as mean±SEM.

Mutant Forms of tmpk Prevent Growth of Transduced K562 Cells Xenografted into AZT-Treated NOD/SCID Mice.

Female or male 5 to 8-week-old non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were purchased from Jackson Laboratory. Lentivirally-transduced or non-transduced K562 cells (20×10⁶ cells) were resuspended in 0.5 mL Dulbecco's phosphate-buffered saline (D-PBS) per inoculum and injected subcutaneously (SC) into the right flanks of recipient mice. AZT treatment, which was administered intraperitoneally (IP) at the dose of 2.5 mg/kg/day, was started one day after injection and conducted for 14 days. In vivo tumor cell growth was monitored by measuring tumor size for up to 32 days post-inoculations. All experimental data were reproduced at least twice.

Transduction of Primay Cultured Human or Mouse T Cells and Analysis of Transgene Expression

Human T lymphocytes are obtained from peripheral blood mononuclear cells (PBMNCs) isolated from heparinized blood obtained from healthy human donors by Ficoll-Hypaque (Amersham-Pharmacia) separations. Mouse T are prepared from the spleen following B cell depletion using goat anti-mouse IgG beads. T cells are activated by using anti-CD3 and anti-CD28 coated beads in a ratio of 1:3 (cell: beads) with 20 IU/mL of recombinant human interleukin 2 for 3 days. Cells were infected with concentrated virus stocks using an MOI of indicated in the presence of 8 μg/ml protamine sulfate. Infected cells were then kept in culture for 5 days prior to evaluating gene transfer efficiency. Gene transfer efficiencies were measured by flow cytometry using a monoclonal anti-human CD19-antibody conjugated with phycoerythrin (PE). About 10⁶ non-transduced and virally transduced cells were incubated for 15 min with the antibody or the corresponding IgG₁ isotype control antibody at 4° C. Cells were washed with phospahate-buffered saline (PBS). Cell analysis was performed on a FACS Calibur and data were analyzed using Cell Quest software.

Statistical Analysis

Statistical analyses was performed using Instat 2.00 (GraphPad). The unpaired Student's t test was used to determine statistical significance. In some experiments, a one-way analysis of variance (ANOVA) with a Bonferroni post-test was used to determine statistically significant results.

Example 2 Generation and Titration of tmpk cDNA Carrying Lentiviral Vectors

Two bi-cistronic lentiviral vectors with either wild-type or mutant human tmpk cDNA located at the upstream of EMCV-IRES sequence and mutant form of human CD19 which was deleted intracellular domain were constructed (FIG. 1). These vectors were derived from LV-EGFP which is a lentiviral vector expressing enhanced GFP under the control of the internal elongation factor-alpha (EF1-a) promoter. The virus titers obtained for each transfer vector were shown in Table 1.

It is known that the expression level of downstream gene by IRES-dependent manner in the bicistronic vector is in between 20 to 50% of that of upstream gene. However, the IRES-dependent expression of downstream gene also depends on the cell-type. While no CD19-expression was seen in transduced-HeLa cells, the expression was detected in the transduced 293T cells. We, however, could detect EGFP expression in the transduced HeLa cells as well as that in 293T cells. These data indicate that when the inventors used the IRES-element for expressing the gene of interests on both upstream and down stream of IRES-sequence in lentiviral system, the inventors need to use 293T cells to measure the functional titer of the virus.

Example 3 Transduction of Jurkat Cells with Recombinant Lentiviruses

To compare the cell killing activity of each LV-constructs expressing tmpk cDNA, the inventors transduced human leukemia cell line Jurkat cells with using an MOI of 10 for 24 hrs. After 5 days of transduction, the inventors tested the CD19 expression in the transduced cells. While no CD19 expression was observed in non-transduced cells, strong CD19-expression was detected on each LV-transduced cells (FIG. 3). The mean fluorescent intensity of CD19 in each LV-transduced cells showed almost same levels indicates that that each LV-transduced cell expressed CD19 in a similar level.

To test the expression levels of the upstream gene in each LV-contruct, the inventors examined Western blot analysis using both rabbit anti-human tmpk as well as rabbit anti-human beta actin as an internal control. Since tmpk is expressed endogenously in the non-transduced Jurkat cells, the inventors could see the tmpk-gene expression in the cells. Comparing the LV-IRES-hCD19-transduced cells and non-transduced cells, LV-tmpk (wild-type; WT)-IRES-hCD19 or LV-tmpk-mutant cDNA-IRES-hCD19-transduced cells showed an increase of tmpk expression in the cells up to 10 times (FIG. 2).

Example 4 Measure AZT-Sensitivity of the Transduced Cells

To examine the AZT-mediated cellkilling activity of tmpk cDNA, each of transduced cells were incubated with the increasing concentration of AZT. After incubating both non-transduced and LV-transduced cells with AZT for 5 days, cell viability was determined using MTT assay (FIG. 4A). These transduced cells were efficiently and selectively killed in a dose-dependent manner by AZT (IC₅₀ of 2 μM), while wild-type tmpk transduced cells were non-sensitive to AZT up to 100 μM. Among of them, both LV-tmpk F105Y and LV-tmpk R16GLL transduced cells showed the dose-dependent cell killing activity. Since MTT assay reflects mitochondrial enzymatic activities in living cell to metabolize the MTT-assay substrate, AZT-metabolites supposed to inhibit mitochondrial function and induced cellular death. To confirm the induction of cellular death such as an apoptosis, the inventors next examined the induction of apoptosis following AZT-treatment in the tmpk-expressing cells by flowcytomeric analysis following the annexin V-staining of the cells. In response to AZT treatment, the early apoptotic cell indices of cells transduced with wild-type tmpk, F105Y or LL were 6.2±0.3%, 40.7±1.7%, and 46.1±4.6%, respectively (n=3). No induction of apoptosis by AZT was observed in the group of negative control group including non-transduced cells and IRES (FIG. 5). In contrast, significant increases in the apoptosis-induced cells were observed in the LV-tmpk transduced cells following AZT-treatment.

Example 5 Intracellular AZT Metabolite Concentration

To evaluate the intracellular concentration of AZT-antimetabolites in the cells, the inventors have established by HPLC. After treatment of the cells transduced with the tmpk LL with AZT, they efficiently convert AZT into the active antimetabolite form, AZT-triphosphate (AZT-TP) (conversion ratio of AZT-TP to AZT MP 11.3 compared to 0.02 in non-transduced cells) (FIG. 6). Conversion of AZT-TP by cells transduced with wild-type tmpk (conversion ratio of 0.10) is only marginally better than the conversion in non-transduced cells (FIG. 6).

Example 6 AZT-Mediated Loss of Mitochondrial Function is Induced by Expression of TMPK-LargeLid

AZT is a potent inhibitor of HIV replication. However, many patients treated with AZT develop toxic mitochondrial myopathy. Long-term AZT treatment has been shown to induce mitochondrial biochemical dysfunction in AIDS patients. In order to prove the mechanism of the induction of cellular apoptosis after AZT-treatment in the tmpk-transduced cells, the inventors measured the membrane potential of mitochondria by analyzing the decrease of the percentage of red-fluorescence in the flow diagram followed by staining the cells with JC-1 reagent. A significant increase in the loss of mitochondrial membrane potential (Δψ) was found to occur in the LV-tmpk R16GLL transduced cells after AZT-treatment in a time dependent manner, however, negative control cell group cells did not increase the percentage of the mitochondrial membrane potential lose cells (FIG. 7).

Example 7 AZT/Tmpk Mediated Cell Killing does not Need Cellular Proliferation

HSV1-tk mediated cell killing requires cellular proliferation for the cytotoxic effect. Here, the inventors have shown proliferation-independent cell killing using mutant tmpk and AZT. Indirubin-metabolites work as cyclin-dependent kinase inhibitors, which function by competing with ATP for binding to the catalytic subunit. They lead to G2/M arrest in many cell lines and G1/S arrest in Jurkat cells. Indirubin-3′-oxime was used to arrest cell cycling, and then transduced cells were treated with AZT. Only 2% of cells transduced with wild-type tmpk were killed, whereas the inventors attained 20% killing of cells transduced with LV-tmpkF105Y-IRES-hCD19 (FIG. 8).

Example 8 In Vivo Tumor Killing Effect Using the Tmpk-Transduced K562-Xenografted NOD/SCID Mouse Model

The inventors next addressed the cell killing ability of the various tmpk mutants in an in vivo tumor model. K562 erythro-leukemia cells were transduced with either wild-type tmpk or the F105Y mutant and injected subcutaneously into NOD/SCID mice. Mice were then treated with 2.5 mg/kg of AZT for the following two-weeks. Non-transduced K562 cells gave rise to tumors of an average 2000 mm² in size at four and a half weeks past injection. Strikingly, while no significant reduction in tumor volume was apparent in AZT-treated mice injected with K562 cells transduced with wild-type tmpk (2000 mm² on average), the inventors have observed a 6 to 20 fold reduction in tumor volume in mice that were injected with K562 cells transduced with the F105Y tmpk mutant following AZT treatment (100-300 mm² final tumor volume consisting primarily of non-transduced K562 cells) (FIG. 9).

Example 9 Transduction of Primary Cultured Human or Mouse T Cells

Primary cultures of human and mouse T cells were transduced with LV constructs containing tmpk cDNAs using an MOI as indicated in FIGS. 10-12. After 6 days of culture, T cells were assessed for their level of EGFP or CD19 expression. While no EGFP or CD19 expression was observed in non-transduced cells, strong EGFP or CD19-expression was detected in each of the LV-transduced cell cultures (FIGS. 10, 11 and 12).

Example 10

The inventors constructed a LV expression system was constructed carrying wild-type or one of two modified forms of tmpk. These engineered tmpk mutants (F105Y and LL) show substantially increased catalytic conversion of AZT compared to wild-type tmpk. Our vector also includes a truncated form of human CD19 (hCD19D), not normally expressed on the T cell lineage, that can be used to enrich and track transduced cells. Highly efficient (95%) transduction of Jurkat cells (human T cell leukemia line) was attained by a single infection with our LVs (MOI of 10). Both LV-tmpk (F105Y)-IRES-hCD19 and LV-tmpk (LL)-IRES-hC19 transduced cells were efficiently and selectively killed in a dose-dependent manner by AZT (IC₅₀ of 2 μM), while wild-type tmpk transduced cells were unaffected by AZT up to 100 μM. In response to AZT treatment, the apoptotic cell indices of cells transduced with wild-type tmpk, F105Y, or LL were 6.2±0.3%, 40.7±1.7%, and 46.1±4.6%, respectively (n=3). The inventors next established by HPLC that cells transduced with a LV encoding a mutant form of tmpk effectively convert AZT into its active anti-metabolite form, AZT-triphosphate (AZT-TP). Intracellular ratio of AZT-TP to AZT-monophosphate (MP) is 11.3 in cells transduced with a LV encoding the LL mutant of tmpk, compared to 0.02 in non-transduced cells and 0.10 in wild-type tmpk transduced cells. Our findings also revealed that following incubation with indirubin-3-oxime, which inhibits cellular proliferation, and AZT treatment, transduced cells were successfully killed. Thus the cytotoxic mechanism differs from HSV1-tk mediated cell killing and is independent of cell proliferation. The inventors also succeeded in the infection of primary mouse and human T cells to over 40% and 70% transduction efficiency, respectively. Lastly, the inventors have shown that in vivo growth of tumor cells transduced with these mutant tmpk LVs was totally inhibited by treatment with AZT. These results demonstrate that our novel suicide gene therapy system has significant potential for many clinical applications.

Example 11 Safety Component of Vectors Used in Gene Therapy

A lentiviral-alpha galactosidase-A GLA)-IRES-tmpk (F105Y) mutant construct is used to transduce the murine myeloid leukemia cell line, C1498. After transduction of the cells with this virus, the congenic recipient GLA-deficient mice will receive the cells by iv-injection. Without prodrug treatment, the host mouse leads to reproducible deathfrom leukemia in a dose-dependence fashion. The host mouse is administered a prodrug. such as AZT. Prodrug treatment results in killing of the responder cells. The enzymatic activity of GLA in the peripheral blood is monitored. The expansion of C1498 cells in the peripheral blood, bone marrow, liver, and spleen of host animals is determined by flow cytometric analysis. Cells are stained for a marker that identifies C1498 cells and not host cells, such as Ly5.1 and for a marker that identifies recipient cells and C1498 cells such as Ly5.2. The survival of mice with or without prodrug-treatment is determined.

Example 12 In Vivo GvHD in Mouse Models

Differentially labeled activated T cells are transplanted into permissive murine hosts. Upon determination of GvHD AZT or other nucleoside analogy is administered. The mouse receiving modified tmpk expressing cells exhibits a reduction of GvHD compared to controls. GvHD is eradicated in the mouse.

Ly5.1-mouse derived T cells and/or Ly5.2-mouse derived T cells will be transduced with LV-tmpk (F105Y)-IRES-hCD19 or LV-IRES-hCD19 as well as LV-EGFP as a control using an MOI of 20.

Host mice, CB6F1 will receive total body irradiation with a single dose of lethal irradiation (11 Gy), and transduced cells with T cell depleted bone-marrow cells prepared from CB6F1 recipient mice will be infused into host recipients (20M cells/mouse, n=10 of each group). Mice will be monitored for clinical GvHD everyday.

The following signs are included into clinical index: weight loss, hunching, activity, fur texture, and skin integrity.

T cell chimerism are determined by flow cytometry after bleeding from the tail vein. Plasma is isolated from the remaining blood and stored at −80° C. for later determination of cytokines.

When chimerism of Ly5.1-derived T cells will go up to over 10%, mice will receive daily ip AZT-injections using a dose of 2.5 mg/kg.

Organs will be isolated and prepared for histology and immunohistochemistry to evaluate the T cell) infiltration in the tissues.

Example 13 Adoptive Transfer of Human T Cells

Activated human T cells are transduced with either a modified tmpk molecule or a control gene. Isolated cells expressing the modified tmpk or control gene are adoptively transfered into permissive murine strains that can accept human xenografts. AZT or other thymidine analog is administered systemically. The number of T cells are determined at various time points to look for evidence of specific killing.

Human Th1 T cell will be transduced with LV-tmpk (F105Y)-IRES-hCD19 or LV-IRES-hCD19 as well as LV-EGFP as a control using an MOI of 20.

Host mice will receive total body irradiation with a single dose of lethal, and transduced cells will be infused into host recipients (20M cells/mouse, n=10 of each group). Mice will be monitored for clinical GvHD everyday. The following signs are included into clinical index: weight loss, hunching, activity, fur texture, and skin integrity.

Human chimerism are determined by flow cytometry after bleeding from the tail vein. Human chimerism is calculated as follows: human chimerism (%)=[huCD3+/(huCD3++mCD45+)]×100. Plasma is isolated from the remaining blood and stored at −80° C. for later determination of human IgGs and cytokines.

When human chimerism will go up to over 10%, mice will receive daily ip AZT-injections using a dose of 2.5 mg/kg.

Organs will be isolated and prepared for histology and immunohistochemistry to evaluate the T cell infiltration in the tissues.

Example 14 Bystander Killing Effects

PC3 cells are transduced using LV-tmpk (wild-type)-IRES-hCD19 or LV-tmpk (F105Y)-IRES-hCD19 and tmpk-overexpressing cells are screened by Western blotting using rabbiit anti-human tmpk antibody. The resultant cells are used for checking the AZT-sensitivity. The cells are split into 96-well plates (2500 cells/well), and expose to AZT for 4 days. Cell viabilty is determined using MTS-reagent. For bystander studies, the tmpk-transduced cells are cocultured with LV-EGFP transduced PC3 cells in 24 well plate (50000 cells/well). After incubation with 100 μM AZT for 4 days cells, the percentage of EGFP-positive cell in each wells are determined by flow cytometry. If the bystander cell killing occur, EGFP-positive cell population treated with AZT show the decrease in their number compared to that without AZT-treatment.

Example 15 Materials and Methods cDNA Cloning of Human CD19 and Construction of LV Shuttle Vector

Total RNA was extracted from the human Burkitt's lymphoma cell line (Raji) using the TRIZOL reagent (Invitrogen, Carlsbad, Calif.). cDNA templates were generated from total RNA by reverse transcription using oligo-dT primer and Superscript II reverse transcriptase (Invitrogen). The cDNA of full-length huCD19 was obtained by PCR using Platinum Hifi Taq DNA polymerase (Invitrogen) and primers CD19 F1 and CD19 R1 described below. The amplified PCR product was directly ligated into the TA-vector, pPCR-script SK (+) (Stratagene, La Jolla, Calif.) to give pPCR-huCD19full. A truncated form of huCD19 (huCD19Δ), which has the extracellular and transmembrane domains but lacks the cytoplasmic domain, was generated by inverse PCR from pPCR-huCD19full using primers CD19 F2 and CD19 R2 (described below), to give pPCR-huCD19Δ. The F2 primer has a complementary sequence to the stop codon just after the end of the transmembrane domain. Following sequence confirmation of the cDNA inserts in pPCR-huCD19Δ, the cDNA fragments were then isolated and subcloned into the EcoRI site of the shuttle vector pSV-IRES that has a sequence for an IRES element from the EMCV, to give pSV-IRES-huCD19Δ. The primer sequences used for subcloning of the human CD19 cDNA were as follows: CD19 F1: 5′-atgccacctcctcgcctcctcttcttcc-3′ (SEQ ID NO:23) and CD19 R1: 5′-tcacctggtgctccaggtgccc-3′ (SEQ ID NO:24). The truncated CD19 construct was made by inverse-PCR using primers CD19 F2: 5′-ccgccaccgcggtggagctccag-3′ (SEQ ID NO:25) and CD19 R2: 5′-ttaaagatgaagaatgcccacaaggg-3′ (SEQ ID NO:26).

Subcloning of Human Tmpk cDNA and Construction of Bicistronic LVs

To subclone the cDNA for wild-type (WT) human tmpk, PBMNCs were isolated from heparinized blood obtained from healthy donors by Ficoll-Hypaque density gradient separations (GE Healthcare Biosciences, Inc. Freiburg, Germany). The WT human tmpk cDNA was amplified by PCR using first strand cDNA generated from PBMNC RNA by the method above. PCR products containing the WT tmpk cDNA were subcloned into pPCR-scriptSK (+) and sequenced. Mutant forms of tmpk, denoted F105Y and R16GLL, were previously generated^(23,24). The cDNAs for the WT and each mutant form of tmpk were first subcloned into a shuttle vector (pSV-IRES-huCD19Δ) to construct bicistronic expression cassettes that allow simultaneous expression a single mRNA strand, encoding the suicide gene and huCD19Δ. The constructs were then each subcloned downstream of the internal EF1α promoter into an HIV-1-based recombinant LV plasmid, pHR′-cPPT-EF-W-SIN²⁷. As a control for the transduction experiments, the inventorsconstructed a pHR′-cPPT-EF-IRES-huCD19Δ-W-SIN LV by subcloning the IRES-huCD19Δ cassette from the pSV-IRES-huCD19Δ plasmid into pHR′-cPPT-EF-W-SIN. In addition, the inventorsused the pHR′-cPPT-EF-enGFP-W-SIN LV³² containing the enhanced GFP (enGFP AKA EGFP) cDNA.

Preparation of High-Titer LV.

Vesicular stomatitis virus glycoprotein (VSV-g)-pseudotyped lentivectors (LVs), including an enGFP marking vector, were generated by transient transfection of 293T cells with a three plasmid system (the aforementioned pHR′ plasmid constructs, the packaging plasmid pCMVΔR8.91, and the VSV-g envelope encoding plasmid pMD.G³² using CaPO₄ precipitation. Viral supernatants were harvested 48 h later, passed through a 0.45 μm filter, and suspended in PBS containing 0.1% (w/v) BSA after ultracentrifugation at 50,000×g for 2 h at 4° C. The concentrated viral supernatants were serially diluted and titered on 293T cells. Transgene expression in transduced cells was assessed 72 h later using a FACS Calibur (BD Biosciences, San Jose, Calif.) following staining of the transduced and control cells with monoclonal mouse anti-human CD19 conjugated with PE (BD Biosciences) or for enGFP expression. Analysis of the data was performed using Cell Quest software (BD Biosciences).

Transduction and Analysis of Transgene Expression by Flow Cytometric Analysis.

Cells of the human T lymphoma cell line, Jurkat, and of the human erythro-leukemic cell line, K562, were maintained in RPMI 1640 supplemented with 10% FBS (CPAA Laboratories, Etobicoke, ON), 100 U/ml of penicillin, and streptomycin to 100 μg/ml (both Sigma, Oakville, ON). Cells were infected with concentrated virus stocks using an MOI of 10 in the presence of 8 μg/ml protamine sulfate. Infected cells were then kept in culture for 5 days prior to evaluating gene transfer efficiency. Gene transfer efficiencies were measured by flow cytometry as described above. Individual clone cell lines were used for all subsequent experiments. They were derived by limiting dilution and selected based on comparable huCD19Δ expression as determined by flow cytometry (above).

To compare the relative expression levels of tmpk, the transduced cells were first fixed with 4% buffered formalin for 15 min then permeabilized by treatment with PBS containing 0.1% Triton X-100 for 10 min. Cells were incubated with 20% normal goat serum for 30 min and then incubated with rabbit anti-human tmpk (diluted 1:500) for 1 h. The cells were further incubated with goat anti-rabbit IgG conjugated to Alexa488 (diluted 1:500, Molecular Probes Inc., Eugene, Oreg.) for 1 h. All incubations were performed at room temperature. Levels of tmpk expressed in the transduced cells were determined by flow cytometry.

HPLC for AZT-Metabolites.

Cells were cultured in the presence of 100 μM AZT for 36 h. 10⁷ cells were homogenized by sonication in 100 μl of 5% (w/v) trichloroacetic acid (TCA). The supernatant was collected after centrifugation at 10,000×g for 15 min at 4° C. TCA was removed by extraction with an equal volume of 20% tri-n-octylamine in pentane. The neutralized aqueous fraction was directly injected into the HPLC machine (Waters, Milford. MA). Separation of AZT and its metabolites were performed on a C18 column (Waters), with a mobile phase composed of 0.2 M phosphate buffer containing 4 mM tetrabutylammonium hydrogen sulfate (pH 7.5) and acetonitrile in the ratio of 97:3 (v/v)⁴⁸ at a flow rate of 1.5 ml/min. The UV absorbance was monitored at 270 nm. Standards for each AZT-metabolite (AZT-MP, AZT-DP, and AZT-TP) were purchased from Moravek Biochemicals (Brea, Calif.). Five million cell equivalents were injected and analyzed in triplicate.

Determination of AZT-Sensitivity of tmpk-Transduced Jurkat Cells.

Transduced Jurkat cells and single-cell clones were seeded in 96 well plates (2×10⁵ cells/well) in 200 μl of the RPMI medium described above with increasing concentrations of AZT (0, 0.1, 1, 10, 100 μM, and 1 mM). The medium was changed daily. After 4 days of culture, cell viability was determined by using Cell Titer 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, Wis.).

For evaluation of the induction of apoptosis, treated Jurkat clonal cells were stained with Annexin V. Briefly, cells were seeded in 24 well plates (10⁶ cells/well) in 1 ml of medium with or without 100 μM AZT. After 4 days of culture, Annexin V staining was performed according to the manufacturer's protocol (Annexin V-APC: BD Pharmingen). For testing whether AZT-mediated cell killing depends on the cellular proliferation, indirubin-3′-monoxime (final concentration 5 μM, Sigma-Aldrich, St. Louis, Mo.) was added simultaneously with 100 μM AZT to the culture.

To simplify comparative studies a relative apoptotic index was calculated. Here data obtained was normalized by dividing results from AZT treated cells in each condition by the results obtained without added AZT. Values were reported as fold increases. Statistical significance between groups was calculated by ANOVA.

Transduction of Primary T Cells with LVs and Evaluation of Induction of Apoptosis Following AZT Exposure

Human T lymphocytes were isolated from PBMNCs contained within heparinized blood obtained from healthy human donors by Ficoll-Hypaque (GE Healthcare) separations. Mouse T cells were prepared from B cell-depleted splenocyte preparations using goat anti-mouse IgG beads (BioMag, Qiagen, Mississauga, ON). T cells were activated by using anti-CD3 and anti-CD28 coated beads (PMID: 12855580) in a ratio of 1:3 (cell: beads) with 20 IU/mL of recombinant human interleukin 2 (R&D Systems, Minneapolis, Minn.) for 3 days. Cells were infected with concentrated virus stocks for 3 h on ice using an indicated MOI in the presence of 8 μg/ml protamine sulfate. Infected cells were then kept in culture for 5 days prior to evaluating gene transfer efficiency. Gene transfer efficiencies were measured by flow cytometry using a monoclonal anti-human CD19-antibody conjugated with phycoerythrin (PE) as described above. Induction of apoptosis following AZT-exposure was evaluated by Annexin V-staining as above.

Measurement of Mitochondrial Inner Membrane Potential and Activation of Caspase-3.

Transduced cells (10⁶) were treated with 100 μM AZT for 4 days or left untreated. To detect changes in the mitochondrial inner membrane potential, the cells were incubated with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1, Molecular Probes Inc.) for 30 min at 37° C., and were then analyzed using a FACS Calibur. The activation of caspase-3 in cells was examined using the FACS Calibur following incubation with an FITC-labeled caspase 3 inhibitor peptide (FITC-DEVD-FMK, Calbiochem, San Diego, Calif.) for 1 hr at 37° C.

Transduced K562 Cells in a NOD/SCID Xenograft Model

Transduced K562 cells were affinity-purified by MACS using magnetic beads conjugated with an anti-human CD19 monoclonal antibody (Miltenyi Biotec Inc., Auburn, Calif.). The purity of the cells following isolation was evaluated by FACS Calibur. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice (5 to 8-weeks old, purchased from Jackson Laboratories, Bar Harbor, Me.) were maintained at the Animal Resource Centre at the Princess Margaret Hospital (Toronto, ON, Canada). The entire animal experimental procedure followed a protocol approved by the Animal Care Committee of the UHN. Experimental groups consisted of male and female NOD/SCID mice injected with 2×10⁷ K562 cells (resuspended in 0.5 mL D-PBS; Oxoid, Basingstoke, England) that were either lentivirally-transduced (n=10 for each LV) or non-transduced (NT) (n=10). Injections were performed subcutaneously (sc) into the dorsal right flanks of recipient mice as previously described⁴⁹. One day after injection of the cells, half of the mice in each group (n=5) began receiving daily AZT injections, administered intraperitoneally (ip) at a dose of 2.5 mg/kg/day for 14 days. Tumor growth was measured by caliper and calculated as 0.5×length×width² (in mm³) for up to 14 days post-inoculations.

Statistical Analysis

Data are presented as the mean±standard error of the mean (SEM) for in vitro experiments and the mean±standard deviation of the mean (SD) for in vivo experiments. Statistical analyses were performed using StatView version 4.5 software for Macintosh (SAS). For in vitro experiments, a one-way analysis of variance (ANOVA) with either a Bonferroni or a Dunnett post-hoc test was used to determine statistically significant results with the level of significance set at P<0.05. Statistical comparison of means was performed by a two-tailed unpaired Student's t test for in vivo experiments.

Results

Synthesis of Novel Suicide LVs Expressing Modified tmpks and Truncated CD19

FIG. 1 shows a schematic structure of the LVs constructed for this study. Jurkat cells were transduced a single time with our recombinant LVs using an MOI of 10. Five days after transduction, CD19 expression on transduced cells was examined. While no CD19 expression was observed on non-transduced (NT) Jurkat cells, over 95% of the cells transduced with each LV showed strong CD19-expression (data not shown). Next, individual cell clones were isolated by flow cytometry and limiting dilution. The mean fluorescent intensity (MFI) of huCD19Δ expressed on isolated clones of cells transduced with each LV showed similar values (data not shown). To compare the expression levels of the upstream tmpk gene on a gross level, transduced cells were also examined by flow cytometry following intracellular immunostaining with rabbit anti-human tmpk. Since tmpk is endogenously expressed in Jurkat cells, the inventorsdetected basal expression of tmpk in NT cells. Cells transduced with LV-tmpk(WT)-IRES-huCD19Δ or either LV-tmpk mutant-IRES-huCD19Δ showed an increase in tmpk expression, up to 5 times higher compared with non-transduced and LV-IRES-huCD19 transduced cells (data not shown).

Determination of the Major Intracellular AZT Metabolites in LV/Tmpk-Transduced Cells

To confirm functionality of the tmpk mutants overexpressed in transduced cells for the metabolic conversion of AZT, the intracellular amounts of AZT-metabolites were measured by reverse-phase HPLC. Following a 36 h incubation with 100 μM AZT, the cells expressing the R16GLL mutant tmpk efficiently converted AZT-MP into AZT-DP and then to the cytotoxically active metabolite form, AZT-TP, whereas the main metabolite that accumulated in the NT-Jurkat cells was AZT-MP (FIG. 2A). Also no significant increases in the accumulation of AZT-TP or induction of cell death in the cells overexpressing WT tmpk itself were observed (data not shown). To compare the effectiveness of the conversion of AZT-MP to its active metabolite, AZT-TP, the ratio of AZT-TP to AZT-MP in each cell population was calculated from the values of the area under curve of each chromatogram. FIG. 6B shows that overexpression of the R16GLL mutant induced a 615-fold increase (P<0.0001) in the AZT-TP/AZT-MP ratio compared to that of the NT cells, the tmpk WT-overexpressing cells, or the LV-IRES-huCD19Δ-transduced cells. SIMILAR DATA WAS ALSO OBTAINED FOR THE F105Y MUTANT. These data indicate that the cells overexpressing this mutant form of tmpk more efficiently converted AZT-MP to AZT-DP, which was subsequently transformed into its active antimetabolite, AZT-TP, likely by cellular nucleotide diphosphate kinase¹².

AZT Sensitivity of tmpk-Transduced Cells

As transduced cells expressing the mutant forms of tmpk revealed differences in intracellular accumulation of AZT-metabolites, the effect of exposure to AZT on cell viability was next measured. Note that by itself, transduction of Jurkat cells with LVs engineering expression of controls or our modified suicide genes and huCD19Δ did not affect their proliferation (data not shown). For the sensitivity experiments the tmpk-expressing cells were incubated with increasing concentrations of AZT, and after four days determined the percentage of living cells using an MTT assay (FIG. 14). Transduced cells expressing the tmpk mutants F105Y or R16GLL were minimally viable upon addition of AZT in a dose-dependent manner (P<0.0001). In contrast, limited cell killing, even at high doses of AZT up to 1 mM, was observed in the negative control cells including: the tmpk WT- and LV-IRES-huCD19Δ-transduced cells as well as the NT Jurkat cells (P values for the tmpk WT, LV-IRES-huCD19Δ-transduced, and NT cells were 0.0677, 0.0426, and 0.1375, respectively).

Since the formation of nuclear apoptotic bodies were observed by DAPI-staining in the tmpk-mutant-expressing cells treated with AZT (data not shown), active metabolites of AZT may have induced cellular death by apoptosis. The induction of apoptosis in the tmpk-expressing cells was examined following AZT treatment, by staining the cells with Annexin V and performing flow cytometric analyses. In response to AZT exposure, early apoptotic indices of cells expressing the F105Y and the R16GLL tmpk mutants were significantly increased (FIG. 15A) compared to those in the absence of AZT treatment (9.5±0.8, and 8.3±0.4-fold increases of apoptotic index by AZT-treatment for F105Y- and R16GLL-expressing cells, respectively).

HSV-tk-mediated cell killing has been reported to require cellular proliferation to demonstrate the cytotoxic effect of the produced anti-metabolites through DNA chain termination⁴⁰. Thus, for these experiments, the cytotoxic events of AZT on tmpk-expressing cells were assessed to see if they were also dependent on cellular proliferation. Transduced cells were cultured with or without 100 μM AZT in the presence of indirubin-3′-monoxime to arrest cell cycle progression. After 4 days incubation with 5 μM indirubin-3′-monoxime in the absence of AZT, the cells showed cell cycle arrest at G2/M-phase (data not shown). By treating the cells with 100 μM AZT in the presence of 5 μM indirubin-3′-monoxime, the apoptotic indices of the F105Y- and R16GLL-expressing cells were still significantly increased (FIG. 15B) compared to those without AZT-treatment (2.3±0.4, and 2.2±0.2-fold increases, respectively). No significant increases were seen in the apoptotic indices of NT cells, WT tmpk-overexpressing cells, or control LV-IRES-huCD19Δ-transduced cells (FIG. 15B). This suggests that the induction of apoptosis by AZT in the tmpk mutant-expressing cells is, in part, independent of their proliferation status.

Transduction and AZT Sensitivity of Primary Human and Mouse T Cells

Primary cultures of human and mouse T cells were transduced with LV tmpk constructs using an MOI of 20. The LV-tmpk (R16GLL) mutant was not used for the transduction of primary T cells since this version contains bacterial tmpk-sequence that could cause an eventual immunogenic response when used in vivo. After 6 days of culture, transduced and control T cells were assessed for their level of huCD19 expression. While very low huCD19 expression was observed in NT cells, huCD19 expression on primary mouse T cells was significantly increased in each of the LV-transduced cultures reaching levels of >50% (FIG. 16A). Likewise, even higher levels of huCD19 expression were observed on productively transduced human T cells reaching levels of >60% (FIG. 16B). These levels are considerable given that expression of downstream genes in bicistronic cassettes may be only 10% or less of upstream gene expression PMID: 10933956. To test the AZT sensitivity of the productively transduced human T cells, the cells were exposed to 100 μM AZT for 4 days and induction of apoptosis was measured by Annexin V staining. Although the early apoptotic indices of primary NT human T cells were increased by AZT exposure at this dose, the apoptotic index of cells expressing the F105Y tmpk mutant was significantly increased (FIG. 16C) compared to those without AZT treatment (4.0±0.3-fold increases; P<0.0001).

Novel Suicide Mechanism Utilized by the tmpk/AZT Axis

AZT is a potent inhibitor of HIV replication. That said, HIV patients treated with AZT sometimes develop toxic mitochondrial myopathy through induction of mitochondrial biochemical dysfunction^(18,20,21). In order to decipher the mechanism of cellular apoptosis induction in the tmpk-mutant-expressing cells following AZT treatment, the mitochondrial inner membrane potential was measured in intact cells. This gives a direct indication of the activity of mitochondrial energy metabolism. For these experiments, a fluorescent probe, JC-1, was used to examine living cells by flow cytometry. JC-1 is a dye that emits a green fluorescence at low mitochondrial membrane potential⁴¹. At higher membrane potentials, JC-1 forms red fluorescence-emitting “J-aggregates”. A significant increase (P<0.0001) in the loss of mitochondrial inner membrane potential occurred in both the F105Y- and the R16GLL-expressing Jurkat cells (FIG. 17A) following 4 days of AZT treatment compared to controls. Negative control cell groups including the NT-, the WT-overexpressing, or the LV-IRES-huCD19Δ-transduced cells treated with AZT did not demonstrate a similar loss of mitochondrial inner membrane potential (FIG. 17A).

Caspase 3 is a key molecule in the cellular apoptosis pathway; loss of mitochondrial inner membrane potential induces caspase 3 activation in cells⁴². Therefore, caspase 3 activation in tmpk mutant-expressing cells treated with AZT was next evaluated. Populations of F105Y- or R16GLL-expressing cells that were treated with AZT showed a significant increase (FIG. 17B) in the percentage of activated caspase 3-positive cells compared to populations of untreated cells (4.6±0.1 and 7.8±0.5-fold increases, respectively). No significant increases in the percentage of cells with activated caspase 3 were seen in the negative controls (NT and LV-IRES-huCD19Δ-transduced cells) following AZT incubation (FIG. 17B). Interestingly, tmpk WT-overexpressing cells that were treated with AZT showed a slight, but significant, increase of the percentage of active caspase 3-positive cells compared to untreated cells. Taken together, our data collectively demonstrates that the mechanism of the induction of apoptosis by AZT in the tmpk-mutant-expressing cells is the activation of caspase 3 resulting from the increase in the loss of the mitochondrial membrane potential, caused by the accumulation of AZT-TP in the cells.

In Vivo Killing of LV Transduced Cells Mediated by AZT

Next killing of the tmpk-mutant-expressing cells in an in vivo tumor model was examined. K562 erythroid leukemia cells were transduced with the LVs that engineered expression of IRES-huCD19Δ, WT tmpk, or a mutant form of the kinase (F105Y or R16GLL). Since the transduction efficiency of the F105Y LV into the K562 cells was fairly modest (68% of cells positive based on observed huCD19 expression; data not shown), these cells were enriched by FAGS using anti-human CD19 conjugated to phycoerythrin (PE). After enrichment, the percentage of CD19-positive K562 cells was over 95% (data not shown). This also confirms the auxiliary utility of huCD19Δ as a cell surface marker enabling immuno-affinity enrichment of transduced cells. Growth characteristics of productively transduced K562 cells were then assessed. Minimal differences in growth of the LV-transduced cells were observed (data not shown). Next, 2×10⁷ transduced K562 cells were injected s.c. into the right flank of NOD/SCID mice. Starting one day after the cell injection, the mice received daily i.p. injections of AZT (2.5 mg/kg/day) or vehicle for two weeks. According to the UHN ACC SOP for humane endpoints, mice were sacrificed when the tumor burden reached ˜1.5 cm³. In animals injected with non-transduced K562 cells, this endpoint occurred within two weeks post-injection. Mice not receiving AZT treatment quickly developed large tumors in a time dependent manner (FIG. 18A). In contrast, the growth of K562 cells transduced with either of the tmpk mutant LVs (F105Y or R16GLL) was strongly inhibited (P=0.0209 and 0.0174, respectively) by daily AZT injection, and the effects were sustained over time (FIG. 18B). No significant tumor growth inhibition by AZT was observed in the LV-tmpk (WT)-IRES-huCD19Δ-transduced, LV-IRES-huCD19Δ-transduced, or the NT-cell injected mice (FIG. 7B18B).

DISCUSSION

Here the inventors have shown that overexpression of rationally-designed mutant forms of human tmpk with improved kinetics significantly reduce cellular viability following AZT treatment both in vitro and in vivo and is useful for treating disease. In addition, these results show that the mechanism of AZT-induced apoptosis is associated with loss of mitochondrial inner membrane potential and activation of caspase 3 in the tmpk-mutant expressing cells. This mechanism provides significant advantages over previous suicide schemas and also allows for killing of non-dividing cells as shown in FIG. 4.

Tmpk is crucial for the activation of a series of prodrugs, including AZT, by catalyzing the second phosphorylation step. It has been shown that this is a rate-limiting step in the activation of AZT¹⁷, resulting in an accumulation of the intermediate metabolite, AZT-MP. AZT was the first effective treatment for AIDS patients¹³⁻¹⁵, however, long-term treatment with AZT has been reported to induce a severe myopathy characterized by structural and functional alterations in mitochondria as a result of accumulation of AZT-MP^(19,20,22). Inhibition of the mitochondrial inner membrane potential has also been found in the muscle mitochondria of long-term AZT-treated rats²¹. The inventors have shown that accumulation of AZT-TP in the tmpk-mutant expressing cells abolished the inner membrane potential of mitochondria (FIG. 17A) and increased the apoptotic-index as a result of the activation of caspase 3 (FIG. 17B). Interestingly, these results revealed that while accumulation of AZT-MP in the tmpk (WT)-overexpressing cells did not affect the mitochondrial function (FIG. 17A), there was a slight induction of apoptosis in these cells mediated by AZT (FIG. 17B).

Another advantage of the application is that it ensures that a high percentage of transduced cells, for example, cells to be transplanted, express the suicide gene. The use of huCD19Δ as a cell-surface marker increases the ratio of gene-modified cells by immuno-affinity enrichment. The contribution of the CD19 cytoplasmic domain in signal transduction has been assessed by others; in vitro by transfecting the cells with a truncated form of the human cDNA⁴³, and in vivo by using CD19-deficient mouse that expresses a transgene encoding the truncated human CD19³⁹. These studies demonstrated that the cytoplasmic domain of CD19 is a crucial for the signaling and for the in vivo function of the CD19/CD21/CD81/Leu-13 complex. This indicates that the truncated form of human CD19 that employed is unlikely to transmit a signal.

Adoptive immunotherapy using T cells is an efficient approach to treat hematological malignancies^(11,34,44-46). GVHD, however, still remains a major problem following non-T cell-depleted allogeneic BMT⁴⁷. In addition to its utility in deleting gene-modified cells if they undergo transformative events, the inventors have shown that it would be advantageous to incorporate an efficient in vivo safety switch that would enable the elimination of gene-modified T cells in the event of GvHD. The drug GCV has been used to deplete HSV-tk-expressing allogeneic lymphocytes following BMT^(34,44). Depletion is not always complete, however, and unwanted host immune responses against cells expressing this foreign enzyme can impair their function and persistence^(10,11). In addition, T cell responses to multiple epitopes of HSV-tk suggests that modification of immunogenic sequences in HSV-tk would likely be ineffective in ablating this reaction¹¹. The use of human gene products as an alternative suicide gene in such situations is less likely to induce an immune response. Furthermore, most BMT patients are on prophylactic GCV to minimize CMV infections, which decreases the broad clinical utility of HSV-tk-based suicide gene therapy.

The inventors showed that the tmpk-mutant expressing Jurkat cells showed an increase in apoptotic index following AZT-treatment in vitro (FIGS. 14 and 15). NOD/SCID mice xenografted with LV-tmpk-mutant-transduced K562 cells (either F105Y or RG16LL) treated with AZT showed the suppression of tumor growth in vivo (FIG. 18). This data shows that the suicide gene methods of the application eliminate unwanted cells in vivo, including cancer cells and allografted T cells.

Example 16 Mammalian Cell CD19Δ/TMPK Fusion Expression Methods Synthesis of Mammalian Expression Vector Engineering Expression of CD19ΔTmpkF105YR200A Fusion Protein

cDNA for CD19Δ and TmpkF105YR200A was amplified from a previously constructed shuttle vector: pSV-TmpkF105YR200A-IRES-CD19Δ.

CD19Δ cDNA was amplified using the following primers: hCD19Δ Forward: 5′-GCTAGAATTCATGCCACCTCCTCGCCTC-3′ (SEQ ID NO:48) and hCD19Δ Reverse: 5′-GCTAGCCGGCAAGATGAAGAATGCCCACAAGG-3′ (SEQ ID NO:49). TmpkF105YR200A-IRES cDNA was amplified using the following primers: Tmpk-IRES Forward: 5′-AAACTGCAGGGATGGCGGCCCGGCGCGG-3′ (SEQ ID NO:50) and Tmpk-IRES Reverse: 5′-GCTCTAGAATCGTGTTTTTCAAAGGAAAACCACGTCC-3′ (SEQ ID NO:51). PCR products were directly ligated into the TA vector pGEM-T Easy (Promega) and sequenced (ACGT Corp.). The following sequence 5′-GCTTACGAATTCTGACGCTAGCCGGCGGGGCTGCAGCATTACATCTAGATACC GTGAGGATCCGTCGCATGCCATCG-3′ (SEQ ID NO:52) was cloned into pGEM-4Z (Promega) using EcoRI and SphI sites to give pGEM-4Z2. TmpkF105YR200A-IRES cDNA was excised from pGEM-T Easy and subcloned into pGEM-4Z2 using PstI and XbaI sites to give pGEM-4Z2-Tmpk-IRES. CD19Δ cDNA was excised from pGEM-T Easy and subcloned into pGEM-4Z2-Tmpk-IRES using EcoRI and NgoMIV sites to give pGEM-4Z2-CD19ΔTmpk-IRES.

CD19ΔTmpkF105YR200A/IRES cDNA was excised from pGEM-4Z2-CD19ΔTmpk-IRES and subcloned into p-CI neo Mammalian Expression Vector (Promega) using EcoRI and XbaI sites to give pCIneo/CD19ΔTmpkF105YR200A/IRES.

Transfection of 293T Cells with p-Clneo/CD19ΔTmpkF105YR200A/IRES

293T cells were transiently transfected with pCIneo/CD19ΔTmpkF105YR200A/IRES using polyethylenimine. 48 hours later, transfection efficiency was determined by staining transfected cells with mouse anti-human CD19 antibody conjugated to phycoerythrin and measuring CD19 expression by FACS analysis.

HPLC for AZT Metabolites

Non-transfected 293T cells and 293T cells transfected with pCIneo/CD19ΔTmpkF105YR200A/IRES were cultured in 100 μM AZT in DMEM media for 6 hours. 1.5×10⁷ cells were harvested and lysed by sonication in 5% (w/v) trichloroacetic acid. The lysate was centrifuged at 10,000 g for 15 minutes at 4° C. Supernatant was collected and trichloroacetic acid was extracted using an equal volume of 20% tri-n-octylamine in pentane. The aqueous fraction was injected into the HPLC machine. A C18 column (Waters) was used to separate AZT metabolites in a mobile phase of 0.2 M phosphate buffer containing 4 mM tetrabutylammonium hydrogen sulfate (pH 7.5) and acetonitrile in a ratio of 97:3 (v/v) at a flow rate of 1.5 ml/min. UV absorbance was measured at 270 nm.

Results HPLC for AZT Metabolites in Transfected 293T Cells

293T cells transfected with pCIneo/CD19ΔTmpkF105YR200A/IRES and cultured in 100 μM AZT for 6 hours showed an increase in the ratio of AZT-DP and AZT-TP to AZT-MP compared to the non-transfected control group (FIG. 19). This data suggests that the CD19ΔTmpkF105YR200A fusion protein is functional and has increased activity towards AZT-MP compared to wild type Tmpk.

Example 17 CD19/Tmpk Fusion Lentivirus Methods Synthesis of Lentiviral Vector Engineering Expression of CD19ΔTmpkF105YR200A Fusion Protein

The CD19ΔTmpkF105YR200A fusion polypeptide was made as described previously. As mentioned, cDNA for CD19Δ and TmpkF105YR200A was amplified from a previously constructed shuttle vector: pSV-TmpkF105YR200A-IRES-CD19Δ. CD19Δ cDNA was amplified using the following primers: hCD19Δ Forward: 5′-GCTAGAATTCATGCCACCTCCTCGCCTC-3′ (SEQ ID NO:48) and hCD19Δ Reverse: 5′-GCTAGCCGGCAAGATGAAGAATGCCCACAAGG-3′ (SEQ ID NO:49). TmpkF105YR200A-IRES cDNA was amplified using the following primers: Tmpk-IRES Forward: 5′-AAACTGCAGGGATGGCGGCCCGGCGCGG-3′ (SEQ ID NO:50) and Tmpk-IRES Reverse: 5′-GCTCTAGAATCGTGTTTTTCAAAGGAAAACCACGTCC-3′ (SEQ ID NO:51). PCR products were directly ligated into the TA vector pGEM-T Easy (Promega) and sequenced (ACGT Corp.). The following sequence 5′-GCTTACGAATTCTGACGCTAGCCGGCGGGGCTGCAGCATTACATCTAGATACC GTGAGGATCCGTCGCATGCCATCG-3′ (SEQ ID NO:52) was cloned into pGEM-4Z (Promega) using EcoRI and SphI to give pGEM-4Z2. TmpkF105YR200A-IRES cDNA was excised from pGEM-T Easy and subcloned into pGEM-4Z2 using PstI and XbaI to give pGEM-4Z2-Tmpk-IRES. CD19Δ cDNA was excised from pGEM-T Easy and subcloned into pGEM-4Z2-Tmpk-IRES using EcoRI and NgoMIV to give pGEM-4Z2-CD19ΔTmpk-IRES. The CD19ΔTmpkF105YR200A fusion cDNA was then amplified from pGEM-4Z2-CD19ΔTmpk-IRES using the following primers: hCD19Δ AscI-Forward: 5′-AGGCGCGCCCGCCACCATGCCACCTCCTCGCCTCCTC-3′ (SEQ ID NO:53) and Tmpk BamHI-Reverse 5′-GCATTACGGGATCCTCACTTCCATAGCTCCCCCAG-3′ (SEQ ID NO:54). PCR product was directly ligated into the TA vector pGEM-T Easy. The CD19ΔTmpkF105YR200A fusion cDNA is then excised from pGEM-T Easy and subcloned into a lentivector backbone that is self inactivating and comprises a cPPT cassette, Elongation Factor 1 alpha promoter [and WPRE sequence such as a pHR′ backbone, to give a lentiviral vector comprising CD19ΔTmpkF105YR200A. The sequence of the final construct is then confirmed.

CD19/Tmpk Fusion Polypeptide

Below is the DNA sequence for the CD19ΔTmpkF105YR200A fusion protein. Letters in green and underlined (N-terminal end) correspond to CD19Δ, letters in blue and not underlined correspond to the linker region (middle), and letters in brown and underlined correspond to TmpkF105YR200A (C-terminus end).

CD19Δ is a truncated form of the full CD19 sequence. The cDNA sequence was truncated at base 939 (amino acid 313), resulting in the deletion of the cytoplasmic region of the protein. Other truncations are also useful.

TmpkF105YR200A sequence also contains two silent mutations:

Base 318 is converted from C to A Base 345 in converted from T to C

These mutations do not alter the sequence of the protein. Accordingly, sequences comprising “C” at base 318 and/or “T” at 345 as well as other nucleotide changes that do not result in amino acid changes, result in only conserved changes, and/or result in changes that are outside a region detected by a CD19 antibody, are also useful.

SEQ ID NO:40 provides a sequece for CD19deltaTmpkF105YR200A polynucleotide. CD19Δ to increase translation of this sequence (not shown above).

Polypeptide Sequence

The amino acid sequence of the CD19ΔTmpkF105YR200A fusion protein is provided in SEQ ID NO:41. A G G A A G represent the linker sequence between CD19Δ and TmpkF105YR200A.

Preparation of High-Titer Lentivirus

Vesicular stomatitis virus glycoprotein pseudotyped lentiviral vectors (LV/CD19ΔTmpkF105YR200A) are produced by transiently co-transfecting 293T cells with pCMVΔ8.91 (packaging plasmid), pMD.G (pseudotyping plasmid) and a lentiviral construct comprising CD19ΔTmpkF105YR200A using polyethylenimine. Viral supernatants are collected 48 hours after transfection, passed through a 0.45 μm filter and concentrated by ultracentrifugation at 28 000 rpm for 90 minutes. To determine functional titer, 293T cells are transduced with serial dilutions of concentrated LV/CD19ΔTmpkF105YR200. Transduced 293T cells are then stained with mouse anti-human CD19 antibody conjugated to phycoerythrin and CD19 expression is measured by FACS analysis.

Determining AZT Sensitivity of Transdced Jurkat Cells

Jurkat cells are transduced with either a LV/IRES/CD19Δ, LV/TmpkF105YR200A/IRES/CD19Δ or LV/CD19ΔTmpkF105YR200A construct. Transduced Jurkat cells and a non-transduced control group are seeded at 2×10⁵ cells in 6-well tissue culture plates. Cells are incubated in the following concentrations of 3′-azido-3′-deoxythymidine (AZT) for 4 days: 0, 0.1, 1.0, 10, 100, 1000 μM AZT. After 4 days cell viability is determined using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega). Data for each group is normalized to the 0 μM AZT value. Samples are assayed in quadruplicate.

Expected Results

Jurkat cells transduced with LV/CD19ΔTmpkF105YR200A are expected to have reduced cell viability compared to the non-transduced control group after incubation for 4 days in 100 μM and 1000 μM AZT. The reduction in cell viability is expected to be comparable to that observed with Jurkat cells transduced with LV/TMPKF105YR200A/IRES/CD19Δ.

Example 18 CD19/Tmpk Fusion Lentivirus Methods Synthesis of Lentiviral Vector Engineering Expression of CD19ΔTmpkF105YR200A Fusion Protein

cDNA for CD19Δ and TmpkF105YR200A was amplified from a previously constructed shuttle vector: pSV-TmpkF105YR200A-IRES-CD19Δ.

CD19Δ cDNA was amplified using the following primers: hCD19Δ Forward: 5′-GCTAGAATTCATGCCACCTCCTCGCCTC-3′ (SEQ ID NO:48) and hCD19Δ Reverse: 5′-GCTAGCCGGCAAGATGAAGAATGCCCACAAGG-3′ (SEQ ID NO:49). TmpkF105YR200A-IRES cDNA was amplified using the following primers: Tmpk-IRES Forward: 5′-AAACTGCAGGGATGGCGGCCCGGCGCGG-3′ (SEQ ID NO:50) and Tmpk-IRES Reverse: 5′-GCTCTAGAATCGTGTTTTTCAAAGGAAAACCACGTCC-3′ (SEQ ID NO:51). PCR products were directly ligated into the TA vector pGEM-T Easy (Promega) and sequenced (ACGT Corp.). The following sequence 5′-GCTTACGAATTCTGACGCTAGCCGGCGGGGCTGCAGCATTACATCTAGATACC GTGAGGATCCGTCGCATGCCATCG-3′ (SEQ ID NO:52) was cloned into pGEM-4Z (Promega) using EcoRI and SphI to give pGEM-4Z2. TmpkF105YR200A-IRES cDNA was excised from pGEM-T Easy and subcloned into pGEM-4Z2 using PstI and XbaI to give pGEM-4Z2-Tmpk-IRES. CD19Δ cDNA was excised from pGEM-T Easy and subcloned into pGEM-4Z2-Tmpk-IRES using EcoRI and NgoMIV to give pGEM-4Z2-CD19ΔTmpk-IRES. The CD19ΔTmpkF105YR200A fusion cDNA was then amplified from pGEM-4Z2-CD19ΔTmpk-IRES using the following primers: hCD19Δ AscI-Forward: 5′-AGGCGCGCCCGCCACCATGCCACCTCCTCGCCTCCTC-3′ (SEQ ID NO:53) and Tmpk BamHI-Reverse 5′-GCATTACGGGATCCTCACTTCCATAGCTCCCCCAG-3′ (SEQ ID NO:54). PCR product was directly ligated into the TA vector pGEM-T Easy. The CD19ΔTmpkF105YR200A fusion cDNA was then excised from pGEM-T Easy and subcloned into a lentivector backbone pCCL.SIN.cPPT.EF.WPRE using AscI and BamHI sites to give pCCL.SIN.cPPT.EF. CD19ΔTmpkF105YR200A.WPRE (FIG. 21). This construct is self inactivating and comprises a central polypurine tract (cPPT), EF and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The sequence of the final construct was then confirmed.

The sequence of the vector construct pCCL.SIN.cPPT.EF. CD19ΔTmpkF105YR200A.WPRE comprising CD19ΔTmpkF105YR200A fusion is provided in SEQ ID NO:42.

The Kozak consensus sequence of cgccacc has been added directly upstream of CD19D to increase translation of this sequence.

Preparation of High-Titer Lentivirus

Vesicular stomatitis virus glycoprotein pseudotyped lentiviral vectors (LV/CD19ΔTmpkF105YR200A) were produced by transiently co-transfecting 293T cells with pCMVΔ8.91 (packaging plasmid), pMD.G (pseudotyping plasmid) and pSIN.cPPT.EF.CD19ΔTmpkF105YR200A.WPRE using polyethylenimine. Viral supernatants were collected 48 hours after transfection, passed through a 0.45 μm filter and concentrated by ultracentrifugation at 28 000 rpm for 90 minutes. To determine functional titer, 293T cells were transduced with serial dilutions of concentrated LV/CD19ΔTmpkF105YR200A. Transduced 293T cells were then stained with mouse anti-human CD19 antibody conjugated to phycoerythrin and CD19 expression was measured by FACS analysis.

Determination of AZT Sensitivity of Transdced Jurkat Cells

Jurkat cells were transduced with either LV/IRES/CD19Δ, LV/TmpkF105YR200A/IRES/CD19Δ or LV/CD19ΔTmpkF105YR200A. Transduced Jurkat cells and a non-transduced control group were seeded at 2×10⁵ cells in 6-well tissue culture plates. Cells were incubated in the following concentrations of 3′-azido-3′-deoxythymidine (AZT) for 4 days: 0, 0.1, 1.0, 10, 100, 1000 μM AZT. After 4 days cell viability was determined using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega). Data for each group was normalized to the 0 μM AZT value. Samples were assayed in quadruplicate.

Results

Jurkat cells transduced with LV/CD19ΔTmpkF105YR200A have significantly reduced cell viability compared to the non-transduced control group after incubation for 4 days in 100 μM and 1000 μM AZT (p<0.001 for both groups) (FIG. 20). The reduction in cell viability is comparable to that observed with Jurkat cells transduced with LV/TMPKF105YR200A/IRES/CD19Δ described above.

Example 19 Cloning of pHR′.cPPT.EF.CD19ΔTmpkF105YR200A.WPRE.SIN

The CD19ΔTmpkF105YR200A fusion cDNA is amplified by PCR from pGEM-4Z2-CD19ΔTmpk-IRES (described previously) using the following primers: hCD19Δ Forward: 5′-GCTAGAATTCATGCCACCTCCTCGCCTC-3′ (SEQ ID NO:48) and Tmpk BamHI-Reverse 5′-GCATTACGGGATCCTCACTTCCATAGCTCCCCCAG-3′ (SEQ ID NO:54). The PCR product is directly ligated into the TA vector pGEM-T Easy. The CD19ΔTmpkF105YR200A fusion cDNA is then excised from pGEM-T Easy and subcloned into pHR′.cPPT.EF.EG.WPRE.SIN using EcoRI and BamHI sites to give pHR′.cPPT.EF.CD19ΔTmpkF105YR200A.WPRE.SIN. It is also possible to use the following pairs of restriction endonucleases to subclone the CD19ΔTmpkF105YR200A fusion cDNA into pHR′.cPPT.EF.EG.WPRE.SIN: AscI and BamHI or SalI and BamHI. If using these restriction endonuclease pairs, the forward PCR primer needs to be designed accordingly to include the correct enzyme sites. A plasmid map for this construct is shown in FIG. 22.

The sequence is provided in SEQ ID NO:43.

Example 20 Jurkat Cells Transduced with LV CD19Δ are Sensitive to AZT

FIG. 23 provides a schematic representation of a CD19Δ/Tmpk construct. Using the mutant Tmpk and by fusing it to a selective marker (CD19) allows for both functional proteins to be expressed by one permitting the use of a monocistronic vector. Also by fusing these two sequences genes together it allows both to be expressed at one to one ratio. Cells express the same number of ΔCD19 and mutant Tmpk functional units. Further, use of the fusion gene decreases the size of the construct.

The CD19 cassette allows selection for modified cells by FACs or beads.

Methods:

Jurkat cells were transduced with pCLL-CD19ΔTmpK and were sorted by FACs staining for CD19. Transduced cells were seeded in 6-well culture plates. Serial dilustion of AZT (Sigma) in media were made from 1000 uM to 0.1 uM and added to each well. Fresh AZT was added daily for 4 days. After 4 days of culture, cells were subjected to the MTS Assay.

Cell Viability Analysis by MTS Assay

Cell viability was determined using the CellTiter 96 AQ One Solution Cell Proliferation Assay (Promega). Absorbance values were read at 490 nm using a plate reader.

Cell viability was reported by normalizing absorbance values at each concentration of AZT against the non-treated value for each group.

Jurkat cells transduced with LV CD19Δ-Tmpk or LV CD19Δ/Tmpk fusion were sensitive to AZT (see FIG. 24).

Example 21 Alpha-Galactosidase A Expression and Activity Methods

Fabry patient Fibroblast and immortalized fabry fibroblasts were transduced with pCCL-CD19ΔTmpk-IRES-αGalA at a MOI 10. Cells were sorted using FACs for 100% CD19 expression.

α-gal A activity was measured in cell lysates of the sorted transduced fibroblast. For details on α-gal A enzyme activity assay see: Yoshimitsu, M. et al. (2007) Gene Therapy. 14, 256-265.

Results:

When considering developing a novel lentiviral vector for gene therapy it needs 3 things:

1. A selective marker. 2. Cell fate control or Suicide gene to protect against insertional mutagenesis. 3. Finally a theurpeutic gene for application of to disease correction.

FIG. 25 shows a schematic representation of a CD19Δ/Tmpk fusion construct with secondary αGALA construct and orientation of the expressed fusion in cells.

The figure shows that the selective marker and the cell fate control or suicide gene are fused together. The fusion allows all cells that express the detection cassette to also express the modified tmpk, which is advantageous.

FIG. 26 shows alphaGalA enzyme activity of transduced Fabry fibroblasts. Codon optimized alphaGalA (CO alphaGalA) was used. CO alpha Gal A, is Codon optimized Alpha Gal A. It's nucleotide sequence is different from the regular Alpha-Gal A. If you align the two sequences (Codon optimized and Normal alphaGalA) you only get about 75% identity, however it you align the protein sequence you get 100% identity. This nucleic acid was synthetically constructed because codon optimization may allow us to get a higher expression for the enzyme because it uses tRNAs that are more abundant within the human cell.

Example 22 Additional Lentiviral Vectors

Two new vectors were sub-cloned from the original pCLL backbone to the pDY lentiviral backbone (note: these vectors have the same functionality as previously described, the functional genes were not altered, they were just moved to a new lentiviral backbone.

The CD19Δ/Tmpk fusion insert was subcloned into a pDY delivery vector (see SEQ ID NO: 44 for vector construct sequence) (see FIG. 27). Codon optimized αGalA (SEQ ID NO: 46; amino acid sequence provided in SEQ ID NO:47) was also cloned into the CD19Δ/Tmpk fusion containing vector (FIG. 28; SEQ ID NO:45).

Construct Names:

pDY-CD19ΔTmpK (SEQ ID NO:44) pDY-CD19ΔTmpK-IRES-00αGalA (codon optimized alpha-Gal A gene) (SEQ ID NO:45)

K562 cells transduced with pDY-CD19ΔTmpK show sensitivity to AZT.

Experimental Design:

K562 cells were transduced with pDY-CD19ΔTmpK (suicide fusion only) at a MOI 10 to examine if this cell line shows sensitivity to AZT.

Transduced cells (only approximately 8% positive for CD19ΔTmpK) were given AZT at concentrations of 0 μM, 0.1 μM, 1.0 μM, 10.0 μM, and 100.0 μM.

Analysis was performed using Flow cytometry after 4 days of AZT treatment (fresh drug was added daily.

Discussion and Results:

See FIG. 29 for results. The Figure shows approximately 80% reduction in transduced cells. If cells were left in culture longer, CD19 expression may have been reduced further.

Control group: GFP positive cells showed no reduction after treatment.

Future experiments will be performed on sorted populations of cells (100% expressing CD19ΔTmpK). Once cells are sorted, a cell proliferation assay (ex. MTS assay) will be used to determine effective killing.

Example 23 Methods

Fabry patient Fibroblast were transduced with pDY-CD19ΔTmpk-IRES-αGalA at a MOI 10. Transduction efficiency was measured by CD19 expression using FAC.

α-gal A activity was measured in cell lysates. For details on α-gal A enzyme activity assay see: Yoshimitsu, M. et al. (2007) Gene Therapy. 14, 256-265.

Results

Transduced Fabry Fibroblast with pDY-CD19ΔTmpK-IRES-COαGalA show enzyme correction (FIG. 30). Fabry primary fibroblasts were infected with the contructs. Data suggest that Fabry cells were corrected even when only approximately 19% cells were transduced with pDY-CD19ΔTmpK-IRES-COaGalA. Enzyme activity was measured in an aGalA enzyme activity assay.

Example 24 Transduced K562 Cells with pDY-CD19ΔTmpK-IRES-COαGalA Show Over-Expression of αGalA Results and Discussion:

Even in K562 cells (normally express aGalA), we are able to increase aGalA levels (1.8 fold increase of MOI 20 compared to untransduced) (FIG. 31). This is an unsorted population of cells (ex. only 3.3% of cells were transduced at a MOI 20). Future experiments will be performed on sorted populations of cells which are expected to show even more striking results.

Example 25

Fabry Disease is an in-born X-linked lysosomal disorder caused by a deficiency of the α-galactosidase A (α-gal A) enzyme. As a result of this deficiency, glycosphingolipids with terminal α-galactosyl moieties accumulate in the vascular endothelium, mainly as globotriaosylceramide (GB3). Progressive GB3 accumulation results in ischemia and infarction, and eventually leads to other major clinical manifestations of the disease, such as kidney and heart failure. Emerging success in gene therapy research has led to possible treatment hopes for Fabry disease. Yoshimitsu and colleagues (2007) (Yoshimitsu, M. et al. (2007) Gene Therapy. 14, 256-265). [2] showed efficient and sustained correction of Fabry mice and patient cells mediated by lentiviral transduction of hematopoietic stem cells. The promises of gene therapy, however, are often impeded by a small number of adverse events as exemplified in past gene therapy clinical trials. In a small number of clinical trials, insertional mutagenesis resulting in oncogenesis has been observed after treatment with integrating retroviral vectors.

One of the safety strategies currently being developed is termed ‘cell fate control’ or suicide gene therapy. Alongside the delivery of a therapeutic gene (ex. α-Gal-A for Fabry disease) a ‘suicide gene’ would also be delivered to gene-knock-in/target cells. Transfer of suicide genes to modified cells endows them with the appropriate enzyme to convert prodrugs to cytotoxic metabolites. This can act as a molecular safety switch in the rare event of insertional oncogenesis. In the event of an adverse effect, the patient can be administered the prodrug for selective clearance of modified cells. Sato et al (2007) (Sato, T., et al. (2007) Molecular Therapy. 15, 962-9) describe a novel enzyme (suicide gene) and prodrug combination for selectively inducing apoptosis in lentiviral vector (LV) transduced cells. The engineered mutant human Thymidylate kinase (tmpk) can efficiently phosophorylate 3′ azido-3′-deoxythymidine (AZT) to its cytotoxic form. Coupling this described suicide gene together in with the therapeutic gene (α-Gal-A) for treatment of Fabry disease in a promising avenue to pursue, it should allow for disease correction with the added benefits of protection against insertional mutagenesis.

Disclosed herein is a novel engineered lentiviral vector containing three components necessary for a gene therapeutic agent. Firstly, a selective marker to enrich for modified cells. Secondly, a ‘suicide gene’ as a safety mechanism. Lastly, the therapeutic gene for disease correction. By creating a fusion protein the integrity of 3 genes could be maintained within a bicistronic vector. Fusing Tmpk to the C-terminus of a truncated from of CD19 (a selective marker), has allowed for the secondary gene, α-gal A (for Fabry disease correction) to be expressed downstream of IRES element, thus creating a bicistronic vector. This novel bicistronic lentiviral vector may be capable of correcting inborn genetic disorders such as Fabry disease and furthermore contains the built-in safety ‘molecular switch,’ required for approving clinical protocols.

Furthermore the suicide fusion vector (CD19ΔTmpk) alone has many additional applications in regards to cell transplantation in the clinic. It may be possible to protect patients from teratomas in stem cell transplantation and Graft-versus-host disease in lymphocyte transfusions. In the event of a cell transplantation adverse event (ex. GvHD or development of a teratoma) it may be possible to eliminate the problematic cells by providing the patient with the prodrug.

Example 26

Once Fabry patients are identified as good candidates for treatment using this propose lentivirus-mediated gene therapy, the patients hematopoietic stem cells will have to be harvested, for ex vivo, genetic manipulation using the lentivirus, pDY-CD19ΔTmpK-IRES-COαGalA. Hematopoietic stem cell isolation will most likely occur from already standardized human clinical protocols such as isolating CD34+ from the blood through the process of apheresis. This procedure is similar to donating blood, the patient is administered certain drugs that stimulate the release of stem cells from the bone marrow into circulating blood. An IV is inserted into the patients arm, and the stem cells are filtered out of the blood.

Once the hematopoietic stem cells (ex. CD34+) are isolated they will be brought to the laboratory were they will be infected with lentivirus, pDY-CD19ΔTmpK-IRES-COαGalA (multiplicity of infection and number of cells unknown at this time). After cells have been successfully infected, the transduction efficiency can be evaluated by the CD19 expression. It is also possible enrich for modified cells (CD19 expressing cells) by FACs, or by using magnetic bead sorting.

The modified hematopoietic stem cells will than be re-introduced into the same patient (autogeneic transplantation). Again already human clinical protocols will be used for cell transplantation and myeloablation regimens of the patient.

After successful cell engraftment (approximately 3 months) and reconstitution of the hematopoietic system the patients α-galactisidase A enzyme activity can be measured in blood plasma to look for correction of Fabry disease (increase levels of enzyme).

In the rare case of an adverse event such as insertional oncogenesis, the patient can be administered the prodrug, 3′ azido-3′-deoxythymidine (AZT), for selective clearance of the modified cells. It should also be noted that AZT is already used in the clinic for treatment of HIV and its safety profiles are well understood.

While the application has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

All publications, patents and patent applications, are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

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1-82. (canceled)
 83. A method of enhancing the capability of a mammalian cell in phosphorylating AZT comprising introducing into the cell a composition comprising: a stably integrating delivery vector; a modified mammalian thymidylate kinase (tmpk) polynucleotide that encodes a modified mammalian tmpk polypeptide that increases phosphorylation of AZT relative to phosphorylation of AZT by wild type mammalian tmpk polypeptide; and a detection cassette polynucleotide that encodes a detection cassette polypeptide that is cell surface expressed, wherein the detection cassette polynucleotide is fused to the modified mammalian tmpk polynucleotide and the detection cassette polypeptide is fused to the modified mammalian tmpk polypeptide.
 84. The method of claim 83, wherein the mammalian cell is selected from the group consisting of a stem cell, an embryonic stem cell, a mesenchymal stem cell, an induced pluripotent stem (IPS) cell, a hematopoietic cell, a T cell and a human cell.
 85. The method of claim 83, further comprising isolating the mammalian cell.
 86. The method of claim 85, wherein the mammalian cell is isolated by contacting the cell with an antibody that binds to the detection cassette polypeptide.
 87. The method of claim 85, wherein the isolated mammalian cell is transplanted into a mammal.
 88. The method of claim 83, wherein (a) the modified mammalian tmpk polypeptide comprises a modification selected from (a) a F to Y mutation at amino acid position 105 of SEQ ID NO: 11, (b) a R to G point mutation at amino acid position 16 of SEQ ID NO: 12, or (c) a R to A mutation at amino acid position 200 of SEQ ID NO: 16 and/or (b) the modified mammalian tmpk polynucleotide comprises a polynucleotide with at least 80% sequence identity to a modified tmpk polynucleotide of any one of SEQ ID NOS: 15, 21, and
 22. 89. The method of claim 83, wherein the modified mammalian tmpk polypeptide further comprises SEQ ID NO: 17 or the amino acid sequence RARGEL.
 90. The method of claim 83, wherein the composition further comprises a therapeutic polynucleotide cassette.
 91. The method of claim 90, wherein the therapeutic polynucleotide cassette is selected from the group consisting of adenosine deaminase, γc interleukin receptor subunit, α-galactosidase A, codon optimized α-galactosidase A, acid ceramidase, galactocerebrosidase, and transmembrane conductance regulator (CFTR) molecules.
 92. A composition comprising: a stably integrating delivery vector; a modified mammalian thymidylate kinase (tmpk) polynucleotide that encodes a modified mammalian tmpk polypeptide that increases phosphorylation of AZT relative to phosphorylation of AZT by wild type mammalian tmpk polypeptide; and a detection cassette polynucleotide that encodes a detection cassette polypeptide that is cell surface expressed, wherein the detection cassette polynucleotide is fused to the modified mammalian tmpk polynucleotide and the detection cassette polypeptide is fused to the modified mammalian tmpk polypeptide.
 93. The composition of claim 92, wherein (a) the modified mammalian tmpk polypeptide comprises a modification selected from (a) a F to Y mutation at amino acid position 105 of SEQ ID NO: 11, (b) a R to G point mutation at amino acid position 16 of SEQ ID NO: 12, or (c) a R to A mutation at amino acid position 200 of SEQ ID NO: 16 and/or (b) the modified mammalian tmpk polynucleotide comprises a polynucleotide with at least 80% sequence identity to a modified tmpk polynucleotide of any one of SEQ ID NOS: 15, 21, and
 22. 94. The composition of claim 92, wherein the modified mammalian tmpk polypeptide further comprises SEQ ID NO: 17 or the amino acid sequence RARGEL.
 95. The composition of claim 92, wherein the delivery vector is selected from the group consisting of a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a spumaviral vector and a plasmid.
 96. The composition of claim 92, wherein the delivery vector is a lentiviral vector that has a pHR′ backbone, a pDY backbone or a pCCL backbone and comprises 5′-Long terminal repeat (LTR), human immunodeficiency virus (HIV) signal sequence, HIV Psi signal 5′-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3′-splice site (SA), Elongation factor (EF) 1-alpha promoter and 3′-Self inactivating LTR (SIN-LTR).
 97. The composition of claim 92, wherein the detection cassette is fused to the tmpk polynucleotide by a linker.
 98. The composition of claim 92, wherein the detection cassette polypeptide fused to the modified mammalian tmpk polypeptide comprises the sequence of SEQ ID NO:38.
 99. The composition of claim 92, wherein the composition comprises a promoter functional in a mammalian cell and the coding sequence of the tmpk/detection cassette fusion polynucleotide is operably connected to the promoter.
 100. The composition of claim 92, wherein the detection cassette polypeptide is EGFR, CD19, truncated CD19, low affinity nerve growth receptor (LNGFR) or truncated LNGFR.
 101. A method of treating Fabry disease comprising administering an isolated cell, isolated virus or composition comprising: a stably integrating delivery vector; a modified mammalian thymidylate kinase (tmpk) polynucleotide that encodes a modified mammalian tmpk polypeptide that increases phosphorylation of AZT relative to phosphorylation of AZT by wild type mammalian tmpk polypeptide; a detection cassette polynucleotide that encodes a detection cassette polypeptide that is cell surface expressed, wherein the detection cassette polynucleotide is fused to the modified mammalian tmpk polynucleotide and the detection cassette polypeptide is fused to the modified mammalian tmpk polypeptide; and a therapeutic polynucleotide cassette selected from α-galactosidase A or codon optimized α-galactosidase A, to a subject in need thereof.
 102. The method of claim 101, wherein the codon optimized α-galactosidase A comprises: (a) SEQ ID NO: 46; (b) SEQ ID NO: 46 wherein T is substituted with U; and/or (c) a sequence with at least 95% identity to SEQ ID NO:46, wherein the sequence is not identical to the polynucleotide having accession number NM_(—)00169. 